Conjugated toxin peptide therapeutic agents

ABSTRACT

Disclosed is a composition of matter comprising an OSK1 peptide analog, and in some embodiments, a pharmaceutically acceptable salt thereof. A pharmaceutical composition comprises the composition and a pharmaceutically acceptable carrier. Also disclosed are DNAs encoding the inventive composition of matter, an expression vector comprising the DNA, and host cells comprising the expression vector. Methods of treating an autoimmune disorder and of preventing or mitigating a relapse of a symptom of multiple sclerosis are also disclosed.

This application claims priority from U.S. Provisional Application No.60/854,674, filed Oct. 25, 2006, and U.S. Application No. 60/995,370,filed Sep. 25, 2007, both of which are hereby incorporated by reference.

This application is related to U.S. Non-provisional application Ser. No.11/978,104, filed Oct. 25, 2007, U.S. Non-provisional application Ser.No. 11/978,105, filed Oct. 25, 2007, U.S. Non-provisional applicationSer. No. 11/978,110 filed Oct. 25, 2007, U.S. Non-provisionalapplication Ser. No. 11/978,111 filed Oct. 25, 2007, and U.S.Non-provisional application Ser. No. 11/978,119, filed Oct. 25, 2007,all which applications are hereby incorporated by reference. Thisapplication is also related to U.S. Non-provisional application Ser. No.11/584,177, filed Oct. 19, 2006, which claims priority from U.S.Provisional Application No. 60/729,083, filed Oct. 21, 2005, both ofwhich applications are hereby incorporated by reference.

The instant application contains an ASCII “txt” compliant sequencelisting submitted via EFS-WEB on May 18, 2010, which serves as both thecomputer readable form (CRF) and the paper copy required by 37 C.F.R.Section 1.821(c) and 1.821(e), and is hereby incorporated by referencein its entirety. The name of the “txt” file created on Sep. 10, 2009,is: A-1186-US-NP-RevSeqList091109.txt, and is 2,607 kB in size.

Throughout this application various publications are referenced withinparentheses or brackets. The disclosures of these publications in theirentireties are hereby incorporated by reference in this application inorder to more fully describe the state of the art to which thisinvention pertains.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is related to the biochemical arts, in particularto therapeutic peptides and conjugates.

2. Discussion of the Related Art

Ion channels are a diverse group of molecules that permit the exchangeof small inorganic ions across membranes. All cells require ion channelsfor function, but this is especially so for excitable cells such asthose present in the nervous system and the heart. The electricalsignals orchestrated by ion channels control the thinking brain, thebeating heart and the contracting muscle. Ion channels play a role inregulating cell volume, and they control a wide variety of signalingprocesses.

The ion channel family includes Na⁺, K⁺, and Ca²⁺ cation and Cl⁻ anionchannels. Collectively, ion channels are distinguished as eitherligand-gated or voltage-gated. Ligand-gated channels include bothextracellular and intracellular ligand-gated channels. The extracellularligand-gated channels include the nicotinic acetylcholine receptor(nAChR), the serotonin (5-hdroxytryptamine, 5-HT) receptors, the glycineand γ-butyric acid receptors (GABA) and the glutamate-activated channelsincluding kanate, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid(AMPA) and N-methyl-D-aspartate receptors (NMDA) receptors. (Harte andOuzounis (2002), FEBS Lett. 514: 129-34). Intracellular ligand gatedchannels include those activated by cyclic nucleotides (e.g. cAMP,cGMP), Ca²⁺ and G-proteins. (Harte and Ouzounis (2002), FEBS Lett. 514:129-34). The voltage-gated ion channels are categorized by theirselectivity for inorganic ion species, including sodium, potassium,calcium and chloride ion channels. (Harte and Ouzounis (2002), FEBSLett. 514: 129-34).

A unified nomenclature for classification of voltage-gated channels wasrecently presented. (Catterall et al. (2000), Pharmacol. Rev. 55: 573-4;Gutman et al. (2000), Pharmacol. Rev. 55, 583-6; Catterall et al. (2000)Pharmacol. Rev. 55: 579-81; Catterall et al. (2000), Pharmacol. Rev. 55:575-8; Hofmann et al. (2000), Pharmacol. Rev. 55: 587-9; Clapham et al.(2000), Pharmacol Rev. 55: 591-6; Chandy (1991), Nature 352: 26; Goldinet al. (2000), Neuron 28: 365-8; Ertel et al. (2000), Neuron 25: 533-5).

The K⁺ channels constitute the largest and best characterized family ofion channels described to date. Potassium channels are subdivided intothree general groups: the 6 transmembrane (6TM) K⁺ channels, the2TM-2TM/leak K⁺ channels and the 2TM/K⁺ inward rectifying channels.(Tang et al. (2004), Ann. Rev. Physiol. 66, 131-159). These three groupsare further subdivided into families based on sequence similarity. Thevoltage-gated K⁺ channels, including (Kv1-6, Kv8-9), EAG, KQT, and Slo(BKCa), are family members of the 6TM group. The 2TM-2TM group comprisesTWIK, TREK, TASK, TRAAK, and THIK, whereas the 2TM/Kir group consists ofKir1-7. Two additional classes of ion channels include the inwardrectifier potassium (IRK) and ATP-gated purinergic (P2X) channels.(Harte and Ouzounis (2002), FEBS Lett. 514: 129-34).

Toxin peptides produced by a variety of organisms have evolved to targetion channels. Snakes, scorpions, spiders, bees, snails and sea anemoneare a few examples of organisms that produce venom that can serve as arich source of small bioactive toxin peptides or “toxins” that potentlyand selectively target ion channels and receptors. In most cases, thesetoxin peptides have evolved as potent antagonists or inhibitors of ionchannels, by binding to the channel pore and physically blocking the ionconduction pathway. In some other cases, as with some of the tarantulatoxin peptides, the peptide is found to antagonize channel function bybinding to a region outside the pore (e.g., the voltage sensor domain).

The toxin peptides are usually between about 20 and about 80 amino acidsin length, contain 2-5 disulfide linkages and form a very compactstructure (see, e.g., FIG. 10). Toxin peptides (e.g., from the venom ofscorpions, sea anemones and cone snails) have been isolated andcharacterized for their impact on ion channels. Such peptides appear tohave evolved from a relatively small number of structural frameworksthat are particularly well suited to addressing the critical issues ofpotency and stability. The majority of scorpion and Conus toxinpeptides, for example, contain 10-40 amino acids and up to fivedisulfide bonds, forming extremely compact and constrained structure(microproteins) often resistant to proteolysis. The conotoxin andscorpion toxin peptides can be divided into a number of superfamiliesbased on their disulfide connections and peptide folds. The solutionstructure of many of these has been determined by NMR spectroscopy,illustrating their compact structure and verifying conservation of theirfamily fold. (E.g., Tudor et al., Ionisation behaviour and solutionproperties of the potassium-channel blocker ShK toxin, Eur. J. Biochem.251(1-2):133-41 (1998); Pennington et al., Role of disulfide bonds inthe structure and potassium channel blocking activity of ShK toxin,Biochem. 38(44): 14549-58 (1999); Jaravine et al., Three-dimensionalstructure of toxin OSK1 from Orthochirus scrobiculosus scorpion venom,Biochem. 36(6):1223-32 (1997); del Rio-Portillo et al.; NMR solutionstructure of Cn12, a novel peptide from the Mexican scorpionCentruroides noxius with a typical beta-toxin sequence but withalpha-like physiological activity, Eur. J. Biochem. 271(12): 2504-16(2004); Prochnicka-Chalufour et al., Solution structure of discrepin, anew K⁺-channel blocking peptide from the alpha-KTx15 subfamily, Biochem.45(6):1795-1804 (2006)).

Conserved disulfide structures can also reflect the individualpharmacological activity of the toxin family. (Nicke et al. (2004), Eur.J. Biochem. 271: 2305-19, Table 1; Adams (1999), Drug Develop. Res. 46:219-34). For example, α-conotoxins have well-defined four cysteine/twodisulfide loop structures (Loughnan, 2004) and inhibit nicotinicacetylcholine receptors. In contrast, ω-conotoxins have sixcysteine/three disulfide loop consensus structures (Nielsen, 2000) andblock calcium channels. Structural subsets of toxins have evolved toinhibit either voltage-gated or calcium-activated potassium channels.FIG. 9 shows that a limited number of conserved disulfide architecturesshared by a variety of venomous animals from bee to snail and scorpionto snake target ion channels. FIG. 7A-7B shows alignment ofalpha-scorpion toxin family and illustrates that a conserved structuralframework is used to derive toxins targeting a vast array of potassiumchannels.

Due to their potent and selective blockade of specific ion channels,toxin peptides have been used for many years as tools to investigate thepharmacology of ion channels. Other than excitable cells and tissuessuch as those present in heart, muscle and brain, ion channels are alsoimportant to non-excitable cells such as immune cells. Accordingly, thepotential therapeutic utility of toxin peptides has been considered fortreating various immune disorders, in particular by inhibition ofpotassium channels such as Kv1.3 and IKCa1 since these channelsindirectly control calcium signaling pathway in lymphocytes. [e.g., Kemet al., ShK toxin compositions and methods of use, U.S. Pat. No.6,077,680; Lebrun et al., Neuropeptides originating in scorpion, U.S.Pat. No. 6,689,749; Beeton et al., Targeting effector memory T cellswith a selective peptide inhibitor of Kv1.3 channels for therapy ofautoimmune diseases, Molec. Pharmacol. 67(4):1369-81 (2005); Mouhat etal., K⁺ channel types targeted by synthetic OSK1, a toxin fromOrthochirus scrobiculosus scorpion venom, Biochem. J. 385:95-104 (2005);Mouhat et al., Pharmacological profiling of Orthochirus scrobiculosustoxin 1 analogs with a trimmed N-terminal domain, Molec. Pharmacol.69:354-62 (2006); Mouhat et al., OsK1 derivatives, WO 2006/002850 A2; B.S. Jensen et al. The Ca²⁺-activated K+ Channel of IntermediateConductance: A Molecular Target for Novel Treatments?, Current DrugTargets 2:401-422 (2001); Rauer et al., Structure-guided Transformationof Charybdotoxin Yields an Analog That Selectively TargetsCa²⁺-activated over Voltage-gated K⁺ Channels, J. Biol. Chem. 275:1201-1208 (2000); Castle et al., Maurotoxin: A Potent Inhibitor ofIntermediate Conductance Ca²⁺-Activated Potassium Channels, MolecularPharmacol. 63: 409-418 (2003); Chandy et al., K⁺ channels as targets forspecific Immunomodulation, Trends in Pharmacol. Sciences 25: 280-289(2004); Lewis & Garcia, Therapeutic Potential of Venom Peptides, Nat.Rev. Drug Discov. 2: 790-802 (2003)].

Small molecules inhibitors of Kv1.3 and IKCa1 potassium channels and themajor calcium entry channel in T cells, CRAC, have also been developedto treat immune disorders [A. Schmitz et al. (2005) Molecul. Pharmacol.68, 1254; K. G. Chandy et al. (2004) TIPS 25, 280; H. Wulff et al.(2001) J. Biol. Chem. 276, 32040; C. Zitt et al. (2004) J. Biol. Chem.279, 12427], but obtaining small molecules with selectivity toward someof these targets has been difficult.

Calcium mobilization in lymphocytes is known to be a critical pathway inactivation of inflammatory responses [M. W. Winslow et al. (2003)Current Opinion Immunol. 15, 299]. Compared to other cells, T cells showa unique sensitivity to increased levels of intracellular calcium andion channels both directly and indirectly control this process. Inositoltriphosphate (IP3) is the natural second messenger which activates thecalcium signaling pathway. IP3 is produced following ligand-inducedactivation of the T cell receptor (TCR) and upon binding to itsintracellular receptor (a channel) causes unloading of intracellularcalcium stores. The endoplasmic reticulum provides one key calciumstore. Thapsigargin, an inhibitor of the sarcoplasmic-endoplasmicreticulum calcium ATPase (SERCA), also causes unloading of intracellularstores and activation of the calcium signaling pathway in lymphocytes.Therefore, thapsigargin can be used as a specific stimulus of thecalcium signaling pathway in T cells. The unloading of intracellularcalcium stores in T cells is known to cause activation of a calciumchannel on the cell surface which allows for influx of calcium fromoutside the cell. This store operated calcium channel (SOCC) on T cellsis referred to as “CRAC” (calcium release activated channel) andsustained influx of calcium through this channel is known to be criticalfor full T cell activation [S. Feske et al. (2005) J. Exp. Med. 202, 651and N. Venkatesh et al. (2004) PNAS 101, 8969]. For many years it hasbeen appreciated that in order to maintain continued calcium influx intoT cells, the cell membrane must remain in a hyperpolarized conditionthrough efflux of potassium ions. In T cells, potassium efflux isaccomplished by the voltage-gated potassium channel Kv1.3 and thecalcium-activated potassium channel IKCa1 [K. G. Chandy et al. (2004)TIPS 25, 280]. These potassium channels therefore indirectly control thecalcium signaling pathway, by allowing for the necessary potassiumefflux that allows for a sustained influx of calcium through CRAC.

Sustained increases in intracellular calcium activate a variety ofpathways in T cells, including those leading to activation of NFAT,NF-kB and AP-1 [Quintana-A (2005) Pflugers Arch.—Eur. J. Physiol. 450,1]. These events lead to various T cell responses including alterationof cell size and membrane organization, activation of cell surfaceeffector molecules, cytokine production and proliferation. Severalcalcium sensing molecules transmit the calcium signal and orchestratethe cellular response. Calmodulin is one molecule that binds calcium,but many others have been identified (M. J. Berridge et al. (2003) Nat.Rev. Mol. Cell. Biol. 4, 517). The calcium-calmodulin dependentphosphatase calcineurin is activated upon sustained increases inintracellular calcium and dephosphorylates cytosolic NFAT.Dephosphorylated NFAT quickly translocates to the nucleus and is widelyaccepted as a critical transcription factor for T cell activation (F.Macian (2005) Nat. Rev. Immunol. 5, 472 and N. Venkatesh et al. (2004)PNAS 101, 8969). Inhibitors of calcineurin, such as cyclosporin A(Neoral, Sandimmune) and FK506 (Tacrolimus) are a main stay fortreatment of severe immune disorders such as those resulting inrejection following solid organ transplant (I. M. Gonzalez-Pinto et al.(2005) Transplant. Proc. 37, 1713 and D. R. J. Kuypers (2005) TransplantInternational 18, 140). Neoral has been approved for the treatment oftransplant rejection, severe rheumatoid arthritis (D. E. Yocum et al.(2000) Rheumatol. 39, 156) and severe psoriasis (J. Koo (1998) BritishJ. Dermatol. 139, 88). Preclinical and clinical data has also beenprovided suggesting calcineurin inhibitors may have utility in treatmentof inflammatory bowel disease (IBD; Baumgart D C (2006) Am. J.Gastroenterol. March 30; Epub ahead of print), multiple sclerosis (Ann.Neurol. (1990) 27, 591) and asthma (S. Rohatagi et al. (2000) J. Clin.Pharmacol. 40, 1211). Lupus represents another disorder that may benefitfrom agents blocking activation of helper T cells. Despite theimportance of calcineurin in regulating NFAT in T cells, calcineurin isalso expressed in other tissues (e.g. kidney) and cyclosporine A & FK506have a narrow safety margin due to mechanism based toxicity. Renaltoxicity and hypertension are common side effects that have limited thepromise of cyclosporine & FK506. Due to concerns regarding toxicity,calcineurin inhibitors are used mostly to treat only severe immunedisease (Bissonnette-R et al. (2006) J. Am. Acad. Dermatol. 54, 472).Kv1.3 inhibitors offer a safer alternative to calcineurin inhibitors forthe treatment of immune disorders. This is because Kv1.3 also operatesto control the calcium signaling pathway in T cells, but does so througha distinct mechanism to that of calcineurin inhibitors, and evidence onKv1.3 expression and function show that Kv1.3 has a more restricted rolein T cell biology relative to calcineurin, which functions also in avariety of non-lymphoid cells and tissues.

Calcium mobilization in immune cells also activates production of thecytokines interleukin 2 (IL-2) and interferon gamma (IFNg) which arecritical mediators of inflammation. IL-2 induces a variety of biologicalresponses ranging from expansion and differentiation of CD4⁺ and CD8⁺ Tcells, to enhancement of proliferation and antibody secretion by Bcells, to activation of NK cells [S. L. Gaffen & K. D. Liu (2004)Cytokine 28, 109]. Secretion of IL-2 occurs quickly following T cellactivation and T cells represent the predominant source of thiscytokine. Shortly following activation, the high affinity IL-2 receptor(IL2-R) is upregulated on T cells endowing them with an ability toproliferate in response to IL-2. T cells, NK cells, B cells andprofessional antigen presenting cells (APCs) can all secrete IFNg uponactivation. T cells represent the principle source of IFNg production inmediating adaptive immune responses, whereas natural killer (NK) cells &APCs are likely an important source during host defense againstinfection [K. Schroder et al. (2004) J. Leukoc. Biol. 75, 163]. IFNg,originally called macrophage-activating factor, upregulates antigenprocessing and presentation by monocytes, macrophages and dendriticcells. IFNg mediates a diverse array of biological activities in manycell types [U. Boehm et al. (1997) Annu. Rev. Immunol. 15, 749]including growth & differentiation, enhancement of NK cell activity andregulation of B cell immunoglobulin production and class switching.

CD40L is another cytokine expressed on activated T cells followingcalcium mobilization and upon binding to its receptor on B cellsprovides critical help allowing for B cell germinal center formation, Bcell differentiation and antibody isotype switching. CD40L-mediatedactivation of CD40 on B cells can induce profound differentiation andclonal expansion of immunoglobulin (Ig) producing B cells [S. Quezada etal. (2004) Annu. Rev. Immunol. 22, 307]. The CD40 receptor can also befound on dendritic cells and CD40L signaling can mediate dendritic cellactivation and differentiation as well. The antigen presenting capacityof B cells and dendritic cells is promoted by CD40L binding, furtherillustrating the broad role of this cytokine in adaptive immunity. Giventhe essential role of CD40 signaling to B cell biology, neutralizingantibodies to CD40L have been examined in preclinical and clinicalstudies for utility in treatment of systemic lupus erythematosis(SLE),—a disorder characterized by deposition of antibody complexes intissues, inflammation and organ damage [J. Yazdany and J Davis (2004)Lupus 13, 377].

Production of toxin peptides is a complex process in venomous organisms,and is an even more complex process synthetically. Due to theirconserved disulfide structures and need for efficient oxidativerefolding, toxin peptides present challenges to synthesis. Althoughtoxin peptides have been used for years as highly selectivepharmacological inhibitors of ion channels, the high cost of synthesisand refolding of the toxin peptides and their short half-life in vivohave impeded the pursuit of these peptides as a therapeutic modality.Far more effort has been expended to identify small molecule inhibitorsas therapeutic antagonists of ion channels, than has been given thetoxin peptides themselves. One exception is the recent approval of thesmall ω-conotoxin MVIIA peptide (Ziconotide™) for treatment ofintractable pain. The synthetic and refolding production process forZiconotide™, however, is costly and only results in a small peptideproduct with a very short half-life in vivo (about 4 hours).

A cost-effective process for producing therapeutics, such as but notlimited to, inhibitors of ion channels, is a desideratum provided by thepresent invention, which involves toxin peptides fused, or otherwisecovalently conjugated to a vehicle.

SUMMARY OF THE INVENTION

The present invention relates to a composition of matter of the formula:(X¹)_(a)-(F¹)_(d)-(X²)_(b)-(F²)_(e)-(X³)_(c)  (I)and multimers thereof, wherein:

F¹ and F² are half-life extending moieties, and d and e are eachindependently 0 or 1, provided that at least one of d and e is 1;

X¹, X², and X³ are each independently -(L)_(f)-P-(L)_(g)-, and f and gare each independently 0 or 1;

P is a toxin peptide of no more than about 80 amino acid residues inlength, comprising at least two intrapeptide disulfide bonds, and atleast one P is an OSK1 peptide analog;

L is an optional linker (present when f=1 and/or g=1); and

a, b, and c are each independently 0 or 1, provided that at least one ofa, b and c is 1.

The present invention thus concerns molecules having variations onFormula I, such as the formulae:P-(L)_(g)-F¹ (i.e., b, c, and e equal to 0);  (II)F¹-(L)_(f)-P (i.e., a, c, and e equal to 0);  (III)P-(L)_(g)-F¹-(L)_(f)-P or (X¹)_(a)-F¹-(X²)_(b) (i.e., c and e equal to0);  (IV)F¹-(L)_(f)-P-(L)_(g)-F² (i.e., a and c equal to 0);  (V)F¹-(L)_(f)-P-(L)_(g)-F²-(L)_(f)-P (i.e., a equal to 0);  (VI)F¹-F²-(L)_(f)-P (i.e., a and b equal to 0);  (VII)P-(L)_(g)-F¹-F² (i.e., b and c equal to 0);  (VIII)P-(L)_(g)-F¹-F²-(L)_(f)-P (i.e., b equal to 0);  (IX)and any multimers of any of these, when stated conventionally with theN-terminus of the peptide(s) on the left. All of such molecules ofFormulae II-IX are within the meaning of Structural Formula I. Withinthe meaning of Formula I, the toxin peptide (P), if more than one ispresent, can be independently the same or different from the OSK1peptide analog, or any other toxin peptide(s) also present in theinventive composition, and the linker moiety ((L)_(f) and/or (L)_(g)),if present, can be independently the same or different from any otherlinker, or linkers, that may be present in the inventive composition.Conjugation of the toxin peptide(s) to the half-life extending moiety,or moieties, can be via the N-terminal and/or C-terminal of the toxinpeptide, or can be intercalary as to its primary amino acid sequence, F¹being linked closer to the toxin peptide's N-terminus than is linked F².Examples of useful half-life extending moieties (F¹ or F²) includeimmunoglobulin Fc domain (e.g., a human immunoglobulin Fc domain,including Fc of IgG1, IgG2, IgG3 or IgG4) or a portion thereof, humanserum albumin (HSA), or poly(ethylene glycol) (PEG). These and otherhalf-life extending moieties described herein are useful, eitherindividually or in combination. A monovalent dimeric Fc-toxin peptidefusion (as represented schematically in FIG. 2B), for example, anFc-OSK1 peptide analog fusion or Fc-ShK peptide analog fusion, is anexample of the inventive composition of matter encompassed by FormulaVII above.

The present invention also relates to a composition of matter, whichincludes, conjugated or unconjugated, a toxin peptide analog of ShK,OSK1, ChTx, or Maurotoxin modified from the native sequences at one ormore amino acid residues, having greater Kv1.3 or IKCa1 antagonistactivity, and/or target selectivity, compared to a ShK, OSK1, orMaurotoxin (MTX) peptides having a native sequence. The toxin peptideanalogs comprise an amino acid sequence selected from any of thefollowing:

SEQ ID NOS: 88, 89, 92, 148 through 200, 548 through 561, 884 through949, or 1295 through 1300 as set forth in Table 2; or

SEQ ID NOS: 980 through 1274, 1303, or 1308 as set forth in Table 7; orany of SEQ ID NOS: 1391 through 4912, 4916, 4920 through 5006, 5009,5010, and 5012 through 5015 as set forth in Table 7A, Table 7B, Table7C, Table 7D, Table 7E, Table 7F, Table 7G, Table 7H, Table 7I or Table7J.

SEQ ID NOS: 330 through 337, 341, 1301, 1302, 1304 through 1307, 1309,1311, 1312, and 1315 through 1336 as set forth in Table 13; or

SEQ ID NOS: 36, 59, 344-346, or 1369 through 1390 as set forth in Table14.

The present invention also relates to other toxin peptide analogs thatcomprise an amino acid sequence selected from, or comprise the aminoacid primary sequence of, any of the following:

SEQ ID NOS: 201 through 225 as set forth in Table 3; or

SEQ ID NOS: 242 through 248 or 250 through 260 as set forth in Table 4;or

SEQ ID NOS: 261 through 275 as set forth in Table 5; or

SEQ ID NOS: 276 through 293 as set forth in Table 6; or

SEQ ID NOS: 299 through 315 as set forth in Table 8; or

SEQ ID NOS: 316 through 318 as set forth in Table 9; or

SEQ ID NO: 319 as set forth in Table 10; or

SEQ ID NO: 327 or 328 as set forth in Table 11; or

SEQ ID NOS: 330 through 337, 341, 1301, 1302, 1304 through 1307, 1309,1311, 1312, or 1315 through 1336 as set forth in Table 13;

SEQ ID NOS: 1369 through 1390 as set forth in Table 14; or

SEQ ID NOS: 348 through 353 as set forth in Table 16; or

SEQ ID NOS: 357 through 362, 364 through 368, 370, 372 through 385, or387 through 398 as set forth in Table 19; or

SEQ ID NOS: 399 through 408 as set forth in Table 20; or

SEQ ID NOS: 410 through 421 as set forth in Table 22; or

SEQ ID NOS: 422, 424, 426, or 428 as set forth in Table 23; or

SEQ ID NOS: 430 through 437 as set forth in Table 24; or

SEQ ID NOS: 438 through 445 as set forth in Table 25; or

SEQ ID NOS: 447, 449, 451, 453, 455, or 457 as set forth in Table 26; or

SEQ ID NOS: 470 through 482 or 484 through 493 as set forth in Table 28;or

SEQ ID NOS: 495 through 506 as set forth in Table 29; or

SEQ ID NOS: 507 through 518 as set forth in Table 30.

The present invention is also directed to a pharmaceutical compositionthat includes the inventive composition of matter and a pharmaceuticallyacceptable carrier.

The compositions of this invention can be prepared by conventionalsynthetic methods, recombinant DNA techniques, or any other methods ofpreparing peptides and fusion proteins well known in the art.Compositions of this invention that have non-peptide portions can besynthesized by conventional organic chemistry reactions, in addition toconventional peptide chemistry reactions when applicable. Thus thepresent invention also relates to DNAs encoding the inventivecompositions and expression vectors and host cells for recombinantexpression.

The primary use contemplated is as therapeutic and/or prophylacticagents. The inventive compositions incorporating the toxin peptide canhave activity and/or ion channel target selectivity comparable to—oreven greater than—the unconjugated peptide.

Accordingly, the present invention includes a method of treating anautoimmune disorder, which involves administering to a patient who hasbeen diagnosed with an autoimmune disorder, such as multiple sclerosis,type 1 diabetes, psoriasis, inflammatory bowel disease (IBD, includingCrohn's Disease and ulcerative colitis), contact-mediated dermatitis,rheumatoid arthritis, psoriatic arthritis, asthma, allergy, restinosis,systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogrensyndrome, inflammatory bone resorption, transplant rejection,graft-versus-host disease, or lupus, a therapeutically effective amountof the inventive composition of matter (preferably comprising a Kv1.3antagonist peptide or IKCa1 antagonist peptide), whereby at least onesymptom of the disorder is alleviated in the patient. In addition, thepresent invention also relates to the use of one or more of theinventive compositions of matter in the manufacture of a medicament forthe treatment or prevention of an autoimmune disorder, such as, but notlimited to, any of the above-listed autoimmune disorders, e.g. multiplesclerosis, type 1 diabetes or IBD.

The present invention is further directed to a method of preventing ormitigating a relapse of a symptom of multiple sclerosis, which methodinvolves administering to a patient, who has previously experienced atleast one symptom of multiple sclerosis, a prophylactically effectiveamount of the inventive composition of matter (preferably comprising aKv1.3 antagonist peptide or IKCa1 antagonist peptide), such that the atleast one symptom of multiple sclerosis is prevented from recurring oris mitigated.

Although mostly contemplated as therapeutic agents, compositions of thisinvention can also be useful in screening for therapeutic or diagnosticagents. For example, one can use an Fc-peptide in an assay employinganti-Fc coated plates. The half-life extending moiety, such as Fc, canmake insoluble peptides soluble and thus useful in a number of assays.

Numerous additional aspects and advantages of the present invention willbecome apparent upon consideration of the figures and detaileddescription of the invention.

U.S. Nonprovisional patent application Ser. No. 11/406,454, filed Apr.17, 2006, is hereby incorporated by reference in its entirety.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows schematic structures of some exemplary Fc dimers that canbe derived from an IgG1 antibody. “Fc” in the figure represents any ofthe Fc variants within the meaning of “Fc domain” herein. “X1” and “X2”represent peptides or linker-peptide combinations as definedhereinafter. The specific dimers are as follows:

FIG. 1A and FIG. 1D: Single disulfide-bonded dimers;

FIG. 1B and FIG. 1E: Doubly disulfide-bonded dimers;

FIG. 1C and FIG. 1F: Noncovalent dimers.

FIG. 2A-C show schematic structures of some embodiments of thecomposition of the invention that shows a single unit of thepharmacologically active toxin peptide. FIG. 2A shows a single chainmolecule and can also represent the DNA construct for the molecule. FIG.2B shows a dimer in which the linker-peptide portion is present on onlyone chain of the dimer (i.e., a “monovalent” dimer). FIG. 2C shows adimer having the peptide portion on both chains. The dimer of FIG. 2Cwill form spontaneously in certain host cells upon expression of a DNAconstruct encoding the single chain shown in FIG. 2A. In other hostcells, the cells could be placed in conditions favoring formation ofdimers or the dimers can be formed in vitro.

FIG. 3A-3B shows exemplary nucleic acid and amino acid sequences (SEQ IDNOS: 1 and 2, respectively) of human IgG1 Fc that is optimized formammalian expression and can be used in this invention.

FIG. 4A-4B shows exemplary nucleic acid and amino acid sequences (SEQ IDNOS: 3 and 4, respectively) of human IgG1 Fc that is optimized forbacterial expression and can be used in this invention.

FIG. 5A shows the amino acid sequence of the mature ShK peptide (SEQ IDNO: 5), which can be encoded for by a nucleic acid sequence containingcodons optimized for expression in mammalian cell, bacteria or yeast.

FIG. 5B shows the three disulfide bonds (—S—S—) formed by the sixcysteines within the ShK peptide (SEQ ID NO: 10).

FIG. 6 shows an alignment of the voltage-gated potassium channelinhibitor Stichodactyla helianthus (ShK) with other closely relatedmembers of the sea anemone toxin family. The sequence of the 35 aminoacid mature ShK toxin (Accession #P29187) isolated from the venom ofStichodactyla helianthus is shown aligned to other closely relatedmembers of the sea anemone family. The consensus sequence and predicteddisulfide linkages are shown, with highly conserved residues beingshaded. The HmK peptide toxin sequence shown (Swiss-Protein Accession#097436) is of the immature precursor from the Magnificent sea anemone(Radianthus magnifica; Heteractis maqnifica) and the putative signalpeptide is underlined. The mature HmK peptide toxin would be predictedto be 35 amino acids in length and span residues 40 through 74. AeK isthe mature peptide toxin, isolated from the venom of the sea anemoneActinia equine (Accession #P81897). The sequence of the mature peptidetoxin AsKS (Accession #Q9TWG1) and BgK (Accession #P29186) isolated fromthe venom of the sea anemone Anemonia sulcata and Bunodosomagranulifera, respectively, are also shown. FIG. 6A shows the amino acidalignment (SEQ ID NO: 10) of ShK to other members of the sea anemonefamily of toxins, HmK (SEQ ID NO: 6 (Mature Peptide), (SEQ ID NO: 542,Signal and Mature Peptide portions)), AeK (SEQ ID NO: 7), AsKs (SEQ IDNO: 8), and BgK (SEQ ID NO: 9). The predicted disulfide linkages areshown and conserved residues are highlighted. (HmK, SEQ ID NO: 543; ShK,SEQ ID NO: 10; AeK, SEQ ID NO: 544; AsKS, SEQ ID NO: 545). FIG. 6B showsa disulfide linkage map for this family having 3 disulfide linkages(C1-C6, C2-C4, C3-C5).

FIG. 7A-7B shows an amino acid alignment of the alpha-scorpion toxinfamily of potassium channel inhibitors. (BmKK1, SEQ ID NO: 11; BmKK4,SEQ ID NO: 12; PBTx1, SEQ ID NO: 14; Tc32, SEQ ID NO: 13; BmKK6, SEQ IDNO: 15; P01, SEQ ID NO: 16; Pi2, SEQ ID NO: 17; Pi3, SEQ ID NO: 18; Pi4,SEQ ID NO: 19; MTX, SEQ ID NO: 20; Pi1, SEQ ID NO: 21; HsTx1, SEQ ID NO:61; AgTx2, SEQ ID NO: 23; KTX1, SEQ ID NO: 24; OSK1, SEQ ID NO: 25;BmKTX, SEQ ID NO: 22; HgTX1, SEQ ID NO: 27; MgTx, SEQ ID NO: 28; C11Tx1,SEQ ID NO: 29; NTX, SEQ ID NO: 30; Tc30, SEQ ID NO: 31; TsTX-Ka, SEQ IDNO: 32; PBTx3, SEQ ID NO: 33; Lqh 15-1, SEQ ID NO: 34; MartenTx, SEQ IDNO: 37; ChTx, SEQ ID NO:36; ChTx-Lq2, SEQ ID NO: 42; IbTx, SEQ ID NO:38; SloTx, SEQ ID NO: 39; BmTx1; SEQ ID NO: 43; BuTx, SEQ ID NO: 41;AmmTx3, SEQ ID NO: 44; AaTX1, SEQ ID NO: 45; BmTX3, SEQ ID NO: 46; Tc1,SEQ ID NO: 48; OSK2, SEQ ID NO: 49; TsK, SEQ ID NO: 54; CoTx1, SEQ IDNO:55; CoTx2, SEQ ID NO: 871; BmPo5, SEQ ID NO: 60; ScyTx, SEQ ID NO:51; P05, SEQ ID NO: 52; Tamapin, SEQ ID NO: 53; and TmTx, SEQ ID NO:691. Highly conserved residues are shaded and a consensus sequence islisted. Subfamilies of the α-KTx are listed and are from Rodriguez de laVega, R. C. et al. (2003) TIPS 24: 222-227. A list of some ion channelsreported to antagonized is listed (IK=IKCa, BK=BKCa, SK=SKCa,Kv=voltage-gated K+ channels). Although most family members in thisalignment represent the mature peptide product, several representimmature or modified forms of the peptide and these include: BmKK1,BmKK4, BmKK6, BmKTX, MartenTx, ChTx, ChTx-Lq2, BmTx1, AaTx1, BmTX3, TsK,CoTx1, BmP05.

FIG. 8 shows an alignment of toxin peptides converted to peptibodies inthis invention (Apamin, SEQ ID NO: 68; HaTx1, SEQ ID NO: 494; ProTx1,SEQ ID NO: 56; PaTx2, SEQ ID NO: 57; ShK[2-35], SEQ ID NO: 92;ShK[1-35], SEQ ID NO: 5; HmK, SEQ ID NO: 6; ChTx (K32E), SEQ ID NO: 59;ChTx, SEQ ID NO: 36; IbTx, SEQ ID NO: 38; OSK1 (E16K, K20D), SEQ ID NO:296; OSK1, SEQ ID NO: 25; AgTx2, SEQ ID NO: 23; KTX1, SEQ ID NO: 24;MgTx, SEQ ID NO: 28; NTX, SEQ ID NO: 30; MTX, SEQ ID NO: 20; Pi2, SEQ IDNO: 17; HsTx1, SEQ ID NO: 61; Anuroctoxin [AnTx], SEQ ID NO: 62; BeKm1,SEQ ID NO: 63; ScyTx, SEQ ID NO: 51; ωGVIA, SEQ ID NO: 64; ωMVIIa, SEQID NO: 65; Ptu1, SEQ ID NO: 66; and CTX, SEQ ID NO: 67). The originalsources of the toxins is indicated, as well as, the number of cysteinesin each. Key ion channels targeted are listed. The alignment showsclustering of toxin peptides based on their source and ion channeltarget impact.

FIG. 9 shows disulfide arrangements within the toxin family. The numberof disulfides and the disulfide bonding order for each subfamily isindicated. A partial list of toxins that fall within each disulfidelinkage category is presented.

FIG. 10 illustrates that solution structures of toxins reveal a compactstructure. Solution structures of native toxins from sea anemone (ShK),scorpion (MgTx, MTX, HsTx1), marine cone snail (wGVIA) and tarantula(HaTx1) indicate the 28 to 39 amino acid peptides all form a compactstructure. The toxins shown have 3 or 4 disulfide linkages and fallwithin 4 of the 6 subfamilies shown in FIG. 9. The solution structuresof native toxins from sea anemone (ShK), scorpion (MgTx, MTX, HsTx1),marine cone snail (wGVIA) and tarantula (HaTx1) were derived fromProtein Data Bank (PDB) accession numbers 1ROO (mmdbld:5247), 1MTX(mmdbld:4064), 1TXM (mmdbld:6201), 1QUZ (mmdbld:36904), 1OMZ(mmdbld:1816) and 1D1H (mmdbld:14344) using the MMDB Entrez 3D-structuredatabase [J. Chen et al. (2003) Nucleic Acids Res. 31, 474] and viewer.

FIG. 11A-C shows the nucleic acid (SEQ ID NO: 69 and SEQ ID NO: 1358)and encoded amino acid (SEQ ID NO:70, SEQ ID NO:1359 and SEQ ID NO:1360) sequences of residues 5131-6660 of pAMG21ampR-Fc-pep. Thesequences of the Fc domain (SEQ ID NOS: 71 and 72) exclude the fiveC-terminal glycine residues. This vector enables production ofpeptibodies in which the peptide-linker portion is at the C-terminus ofthe Fc domain.

FIG. 11D shows a circle diagram of a peptibody bacterial expressionvector pAMG21ampR-Fc-pep having a chloramphenicol acetyltransferase gene(cat; “CmR” site) that is replaced with the peptide-linker sequence.

FIG. 12A-C shows the nucleic acid (SEQ ID NO: 73 and SEQ ID NO: 1361)and encoded amino acid (SEQ ID NO:74, SEQ ID NO: 1362 and SEQ ID NO:1363) sequences of residues 5131-6319 of pAMG21ampR-Pep-Fc. Thesequences of the Fc domain (SEQ ID NOS: 75 and 76) exclude the fiveN-terminal glycine residues. This vector enables production ofpeptibodies in which the peptide-linker portion is at the N-terminus ofthe Fc domain.

FIG. 12D shows a circle diagram of a peptibody bacterial expressionvector having a zeocin resistance (ble; “ZeoR”) site that is replacedwith the peptide-linker sequence.

FIG. 12E-G shows the nucleic acid (SEQ ID NO:1339) and encoded aminoacid sequences of pAMG21ampR-Pep-Fc (SEQ ID NO:1340, SEQ ID NO:1341, andSEQ ID NO:1342). The sequences of the Fc domain (SEQ ID NOS: 75 and 76)exclude the five N-terminal glycine residues. This vector enablesproduction of peptibodies in which the peptide-linker portion is at theN-terminus of the Fc domain.

FIG. 13A is a circle diagram of mammalian expression vector pCDNA3.1(+)CMVi.

FIG. 13B is a circle diagram of mammalian expression vector pCDNA3.1 (+)CMVi-Fc-2×G4S-Activin RIIb that contains a Fc region from human IgG1, a10 amino acid linker and the activin RIIb gene.

FIG. 13C is a circle diagram of the CHO expression vector pDSRa22containing the Fc-L10-ShK[2-35] coding sequence.

FIG. 14A-14B shows the nucleotide and encoded amino acid sequences (SEQ.ID. NOS: 77 and 78, respectively) of the molecule identified as“Fc-L10-ShK[1-35]” in Example 1 hereinafter. The L10 linker amino acidsequence (SEQ ID NO: 79) is underlined.

FIG. 15A-15B shows the nucleotide and encoded amino acid sequences (SEQ.ID. NOS: 80 and 81, respectively) of the molecule identified as“Fc-L10-ShK[2-35]” in Example 2 hereinafter. The same L10 linker aminoacid sequence (SEQ ID NO: 79) as used in Fc-L10-ShK[1-35] (FIG. 14A-14B)is underlined.

FIG. 16A-16B shows the nucleotide and encoded amino acid sequences (SEQ.ID. NOS: 82 and 83, respectively) of the molecule identified as“Fc-L25-ShK[2-35]” in Example 2 hereinafter. The L25 linker amino acidsequence (SEQ ID NO: 84) is underlined.

FIG. 17 shows a scheme for N-terminal PEGylation of ShK peptide (SEQ IDNO: 5 and SEQ ID NO:10) by reductive amination, which is also describedin Example 32 hereinafter.

FIG. 18 shows a scheme for N-terminal PEGylation of ShK peptide (SEQ IDNO: 5 and SEQ ID NO:10) via amide formation, which is also described inExample 34 hereinafter.

FIG. 19 shows a scheme for N-terminal PEGylation of ShK peptide (SEQ IDNO: 5 and SEQ ID NO:10) by chemoselective oxime formation, which is alsodescribed in Example 33 hereinafter.

FIG. 20A shows a reversed-phase HPLC analysis at 214 nm and FIG. 20Bshows electrospray mass analysis of folded ShK[2-35], also described asfolded-“Des-Arg1-ShK” (Peptide 2).

FIG. 21 shows reversed phase HPLC analysis at 214 nm of N-terminallyPEGylated ShK[2-35], also referred to as N-TerminallyPEGylated-“Des-Arg1-ShK”.

FIG. 22A shows a reversed-phase HPLC analysis at 214 nm of foldedShK[1-35], also referred to as “ShK”.

FIG. 22B shows electrospray mass analysis of folded ShK[1-35], alsoreferred to as “ShK”.

FIG. 23 illustrates a scheme for N-terminal PEGylation of ShK[2-35] (SEQID NO: 92 and SEQ ID NO: 58, also referred to as “Des-Arg1-ShK” or “ShKd1”) by reductive amination, which is also described in Example 31hereinafter.

FIG. 24A shows a western blot of conditioned medium from HEK 293 cellstransiently transfected with Fc-L10-ShK[1-35]. Lane 1: molecular weightmarkers; Lane 2: 15 μl Fc-L10-ShK; Lane 3: 10 μl Fc-L10-ShK; Lane 4: 5μl Fc-L10-ShK; Lane 5; molecular weight markers; Lane 6: blank; Lane 7:15 μl No DNA control; Lane 8: 10 μl No DNA control; Lane 9: 5 μl No DNAcontrol; Lane 10; molecular weight markers.

FIG. 24B shows a western blot of with 15 μl of conditioned medium fromclones of Chinese Hamster Ovary (CHO) cells stably transfected withFc-L-ShK[1-35]. Lanes 1-15 were loaded as follows: blank, BB6, molecularweight markers, BB5, BB4, BB3, BB2, BB1, blank, BD6, BD5, molecularweight markers, BD4, BD3, BD2.

FIG. 25A shows a western blot of a non-reducing SDS-PAGE gel containingconditioned medium from 293T cells transiently transfected withFc-L-SmIIIA.

FIG. 25B shows a western blot of a reducing SDS-PAGE gel containingconditioned medium from 293T cells transiently transfected withFc-L-SmIIIA.

FIG. 26A shows a Spectral scan of 10 μl purified bivalent dimericFc-L10-ShK[1-35] product from stably transfected CHO cells diluted in700 μl PBS (blanking buffer) using a Hewlett Packard 8453spectrophotometer and a 1-cm path length quartz cuvette.

FIG. 26B shows Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final bivalent dimeric Fc-L10-ShK[1-35] product. Lanes1-12 were loaded as follows: Novex Mark12 wide range protein standards,0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10μg product non-reduced, Novex Mark12 wide range protein standards, 0.5μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μgproduct reduced.

FIG. 26C shows size exclusion chromatography on 20 μg of the finalbivalent dimeric Fc-L10-ShK[1-35] product injected on to a PhenomenexBioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, andpH 6.9 at 1 ml/min observing the absorbance at 280 nm.

FIG. 26D shows a MALDI mass spectral analysis of the final sample ofbivalent dimeric Fc-L10-ShK[1-35] analyzed using a Voyager DE-RPtime-of-flight mass spectrometer equipped with a nitrogen laser (337 nm,3 ns pulse). The positive ion/linear mode was used, with an acceleratingvoltage of 25 kV. Each spectrum was produced by accumulating data from˜200 laser shots. External mass calibration was accomplished usingpurified proteins of known molecular masses.

FIG. 26E shows Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final monovalent dimeric Fc-L10-ShK[1-35] product. Lanes1-12 were loaded as follows: Novex Mark12 wide range protein standards(10 μL), 0.5 μg product non-reduced (1.3 μL), blank, 2.0 μg productnon-reduced (5 μL), blank, 10 μg product non-reduced (25 μL), NovexMark12 wide range protein standards (10 μL), 0.5 μg product reduced (1.3μL), blank, 2.0 μg product reduced (5 μL), blank, and 10 μg productreduced (25 μL).

FIG. 26F shows size exclusion chromatography on 20 μg of the finalmonovalent dimeric Fc-L10-ShK[1-35] product injected on to a PhenomenexBioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, andpH 6.9 at 1 ml/min observing the absorbance at 280 nm.

FIG. 27A shows a Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final purified bivalent dimeric Fc-L10-ShK[2-35] productfrom stably transfected CHO cells. Lane 1-12 were loaded as follows:Novex Mark12 wide range protein standards, 0.5 μg product non-reduced,blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced,Novex Mark12 wide range protein standards, 0.5 μg product reduced,blank, 2.0 μg product reduced, blank, and 10 μg product reduced.

FIG. 27B shows size exclusion chromatography on 50 μg of the purifiedbivalent dimeric Fc-L10-ShK[2-35] injected on to a Phenomenex BioSep SEC3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, and pH 6.9 at 1ml/min observing the absorbance at 280 nm.

FIG. 27C shows a Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of bivalent dimeric Fc-L5-ShK[2-35] purified from stablytransfected CHO cells. Lane 1-12 are loaded as follows: Novex Mark12wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μgproduct non-reduced, blank, 10 μg product non-reduced, Novex Mark12 widerange protein standards, 0.5 μg product reduced, blank, 2.0 μg productreduced, blank, and 10 μg product reduced.

FIG. 27D shows a Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of bivalent dimeric Fc-L25-ShK[2-35] purified from stablytransfected CHO cells. Lane 1-12 are loaded as follows: Novex Mark12wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μgproduct non-reduced, blank, 10 μg product non-reduced, Novex Mark12 widerange protein standards, 0.5 μg product reduced, blank, 2.0 μg productreduced, blank, and 10 μg product reduced.

FIG. 27E shows a spectral scan of 10 μl of the bivalent dimericFc-L10-ShK[2-35] product diluted in 700 μl PBS (blanking buffer) using aHewlett Packard 8453 spectrophotometer and a 1 cm path length quartzcuvette.

FIG. 27F shows a MALDI mass spectral analysis of the final sample ofbivalent dimeric Fc-L110-ShK[2-35] analyzed using a Voyager DE-RPtime-of-flight mass spectrometer equipped with a nitrogen laser (337 nm,3 ns pulse). The positive ion/linear mode was used, with an acceleratingvoltage of 25 kV. Each spectrum was produced by accumulating data fromabout 200 laser shots. External mass calibration was accomplished usingpurified proteins of known molecular masses.

FIG. 27G shows a spectral scan of 10 μl of the bivalent dimericFc-L5-ShK[2-35] product diluted in 700 μl PBS (blanking buffer) using aHewlett Packard 8453 spectrophotometer and a 1 cm path length quartzcuvette.

FIG. 27H shows the size exclusion chromatography on 50 mg of the finalbivalent dimeric Fc-L5-ShK[2-35] product injected on to a PhenomenexBioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH6.9 at 1 ml/min observing the absorbance at 280 nm.

FIG. 27I shows a MALDI mass spectral analysis of the final sample ofbivalent dimeric Fc-L5-ShK[2-35] analyzed using a Voyager DE-RPtime-of-flight mass spectrometer equipped with a nitrogen laser (337 nm,3 ns pulse). The positive ion/linear mode was used, with an acceleratingvoltage of 25 kV. Each spectrum was produced by accumulating data from˜200 laser shots. External mass calibration was accomplished usingpurified proteins of known molecular masses.

FIG. 27J shows a Spectral scan of 20 μl of the product diluted in 700 μlPBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer anda 1 cm path length quartz cuvette.

FIG. 27K shows the size exclusion chromatography on 50 μg of the finalbivalent dimeric Fc-L25-ShK[2-35] product injected on to a PhenomenexBioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH6.9 at 1 ml/min observing the absorbance at 280 nm.

FIG. 27L shows a MALDI mass spectral analysis of the final sample ofbivalent dimeric Fc-L25-ShK[2-35] analyzed using a Voyager DE-RPtime-of-flight mass spectrometer equipped with a nitrogen laser (337 nm,3 ns pulse). The positive ion/linear mode was used, with an acceleratingvoltage of 25 kV. Each spectrum was produced by accumulating data fromabout 200 laser shots. External mass calibration was accomplished usingpurified proteins of known molecular masses.

FIG. 28A shows a Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of Fc-L10-KTX1 purified and refolded from bacterial cells. Lane1-12 are loaded as follows: Novex Mark12 wide range protein standards,0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10μg product non-reduced, Novex Mark12 wide range protein standards, 0.5μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μgproduct reduced.

FIG. 28B shows size exclusion chromatography on 45 μg of purifiedFc-L10-KTX1 injected on to a Phenomenex BioSep SEC 3000 column (7.8×300mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing theabsorbance at 280 nm.

FIG. 28C shows a Spectral scan of 20 μl of the Fc-L10-KTX1 productdiluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 28D shows a MALDI mass spectral analysis of the final sample ofFc-L10-KTX1 analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses.

FIG. 29A shows a Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of Fc-L-AgTx2 purified and refolded from bacterial cells. Lane1-12 are loaded as follows: Novex Mark12 wide range protein standards,0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10μg product non-reduced, Novex Mark12 wide range protein standards, 0.5μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μgproduct reduced.

FIG. 29B shows a Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of Fc-L10-HaTx1 purified and refolded from bacterial cells.Lane 1-12 are loaded as follows: Novex Mark12 wide range proteinstandards, 0.5 μg product non-reduced, blank, 2.0 μg productnon-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide rangeprotein standards, 0.5 μg product reduced, blank, 2.0 μg productreduced, blank, and 10 μg product reduced, spectral scan of the purifiedmaterial.

FIG. 29C shows a Spectral scan of 20 μl of the Fc-L10-AgTx2 productdiluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 29D shows the Size exclusion chromatography on 20 μg of the finalFc-L10-AgTx2 product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm.

FIG. 29E shows a MALDI mass spectral analysis of the final sample ofFc-L10-AgTx2 analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from about 200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses.

FIG. 29F shows a Spectral scan of 20 μl of the Fc-L10-HaTx1 productdiluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 29G shows the size exclusion chromatography on 20 μg of the finalFc-L10-HaTx1 product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm.

FIG. 29H shows a MALDI mass spectral analysis of the final sample ofFc-L10-HaTx1 analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses.

FIG. 30A shows Fc-L10-ShK[1-35] purified from CHO cells produces aconcentration dependent block of the outward potassium current recordedfrom HEK293 cell stably expressing the human Kv1.3 channel.

FIG. 30B shows the time course of potassium current block byFc-L10-ShK[1-35] at various concentrations. The IC₅₀ was estimated to be15±2 pM (n=4 cells).

FIG. 30C shows synthetic ShK[1-35] (also referred to as “ShK” alone)produces a concentration dependent block of the outward potassiumcurrent recorded from HEK293 cell stably expressing human Kv1.3 channel.

FIG. 30D shows the time course of ShK[1-35] block at variousconcentrations. The IC₅₀ for ShK was estimated to be 12±1 pM (n=4cells).

FIG. 31A shows synthetic peptide analog ShK[2-35] producing aconcentration dependent block of the outward potassium current asrecorded from HEK293 cells stably expressing human Kv1.3 channel with anIC50 of 49±5 pM (n=3 cells).

FIG. 31B shows the CHO-derived Fc-L10-ShK[2-35] peptibody producing aconcentration dependent block of the outward potassium current asrecorded from HEK293 cell stably expressing human Kv1.3 channel with anIC50 of 115±18 pM (n=3 cells).

FIG. 31C shows the Fc-L5-ShK[2-35] peptibody produces a concentrationdependent block of the outward potassium current recorded from HEK293cell stably expressing human Kv1.3 channel with an IC50 of 100 pM (n=3cells).

FIG. 32A shows Fc-L-KTX1 peptibody purified from bacterial cellsproducing a concentration dependent block of the outward potassiumcurrent as recorded from HEK293 cell stably expressing human Kv1.3channel.

FIG. 32B shows the time course of potassium current block by Fc-L10-KTX1at various concentrations.

FIG. 33 shows by immunohistochemistry that CHO-derived Fc-L10-ShK[1-35]peptibody stains HEK 293 cells stably transfected with human Kv1.3 (FIG.33A), whereas untransfected HEK 293 cells are not stained with thepeptibody (FIG. 33B).

FIG. 34 shows results of an enzyme-immunoassay using fixed HEK 293 cellsstably transfected with human Kv1.3. FIG. 34A shows the CHO-derivedFc-L10-ShK[1-35] (referred to here simply as “Fc-L10-ShK”) peptibodyshows a dose-dependent increase in response, whereas the CHO-Fc control(“Fc control”) does not. FIG. 34B shows the Fc-L10-ShK[1-35] peptibody(referred to here as “Fc-ShK”) does not elicit a response fromuntransfected HEK 293 cells using similar conditions and also showsother negative controls.

FIG. 35 shows the CHO-derived Fc-L10-ShK[1-35] peptibody shows adose-dependent inhibition of IL-2 (FIG. 35A) and IFNγ (FIG. 35B)production from PMA and αCD3 antibody stimulated human PBMCs. Thepeptibody shows a novel pharmacology exhibiting a complete inhibition ofthe response, whereas the synthetic ShK[1-35] peptide alone shows only apartial inhibition.

FIG. 36 shows the mammalian-derived Fc-L10-ShK[1-35] peptibody inhibitsT cell proliferation (³H-thymidine incorporation) in human PBMCs fromtwo normal donors stimulated with antibodies to CD3 and CD28. FIG. 36Ashows the response of donor 1 and FIG. 36B the response of donor 2.Pre-incubation with the anti-CD32 (FcgRII) blocking antibody did notalter the sensitivity toward the peptibody.

FIG. 37 shows the purified CHO-derived Fc-L10-ShK[1-35] peptibody causesa dose-dependent inhibition of IL-2 (FIG. 37A) and IFNγ (FIG. 37B)production from αCD3 and αCD28 antibody stimulated human PBMCs.

FIG. 38A shows the PEGylated ShK[2-35] synthetic peptide produces aconcentration dependent block of the outward potassium current recordedfrom HEK293 cell stably expressing human Kv1.3 channel and the timecourse of potassium current block at various concentrations is shown inFIG. 38B.

FIG. 39A shows a spectral scan of 50 μl of the Fc-L10-ShK(1-35) productdiluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 39B shows a Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final Fc-L10-ShK(1-35) product. Lane 1-12 are loaded asfollows: Novex Mark12 wide range protein standards, 0.5 μg productnon-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg productnon-reduced, Novex Mark12 wide range protein standards, 0.5 μg productreduced, blank, 2.0 μg product reduced, blank, and 10 μg productreduced.

FIG. 39C shows the Size exclusion chromatography on 50 μg of the finalFc-L10-ShK(1-35) product injected on to a Phenomenex BioSep SEC 3000column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/minobserving the absorbance at 280 nm.

FIG. 40A shows a Spectral scan of 20 μl of the Fc-L10-ShK(2-35) productdiluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 40B shows a Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final Fc-L10-ShK(2-35) product. Lanes 1-12 are loaded asfollows: Novex Mark12 wide range protein standards, 0.5 μg productnon-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg productnon-reduced, Novex Mark12 wide range protein standards, 0.5 μg productreduced, blank, 2.0 μg product reduced, blank, and 10 μg productreduced.

FIG. 40C shows the size exclusion chromatography on 50 μg of the finalFc-L10-ShK(2-35) product injected on to a Phenomenex BioSep SEC 3000column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/minobserving the absorbance at 280 nm.

FIG. 40D shows a MALDI mass spectral analysis of the final sample ofFc-L10-ShK(2-35) analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses.

FIG. 41A shows spectral scan of 50 μl of the Fc-L10-OSK1 product dilutedin 700 μl Formulation Buffer using a Hewlett Packard 8453spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 41B shows Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final Fc-L10-OSK1 product. Lanes 1-12 are loaded asfollows: Novex Mark12 wide range protein standards, 0.5 μg productnon-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg productnon-reduced, Novex Mark12 wide range protein standards, 0.5 μg productreduced, blank, 2.0 μg product reduced, blank, and 10 μg productreduced.

FIG. 41C shows size exclusion chromatography on 123 μg of the finalFc-L10-OSK1 product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm.

FIG. 41D shows liquid chromatography—mass spectral analysis ofapproximately 4 μg of the final Fc-L110-OSK1 sample using a Vydac C₄column with part of the effluent directed into a LCQ ion trap massspectrometer. The mass spectrum was deconvoluted using the Bioworkssoftware provided by the mass spectrometer manufacturer.

FIG. 42A-B shows nucleotide and amino acid sequences (SEQ ID NO: 1040and SEQ ID NO: 1041, respectively) of Fc-L10-OSK1.

FIG. 43A-B shows nucleotide and amino acid sequences (SEQ ID NO: 1042and SEQ ID NO: 1043, respectively) of Fc-L10-OSK1[K7S].

FIG. 44A-B shows nucleotide and amino acid sequences (SEQ ID NO: 1044and SEQ ID NO: 1045, respectively) of Fc-L10-OSK1[E16K,K20D].

FIG. 45A-B shows nucleotide and amino acid sequences (SEQ ID NO: 1046and SEQ ID NO: 1047, respectively) of Fc-L10-OSK1[K7S,E16K,K20D].

FIG. 46 shows a Western blot (from tris-glycine 4-20% SDS-PAGE) withanti-human Fc antibodies. Lanes 1-6 were loaded as follows: 15 μl ofFc-L10-OSK1[K7S,E16K,K20D]; 15 μl of Fc-L10-OSK1[E16K,K20D]; 15 μl ofFc-L10-OSK1[K7S]; 15 μl of Fc-L10-OSK1; 15 μl of “No DNA” control;molecular weight markers.

FIG. 47 shows a Western blot (from tris-glycine 4-20% SDS-PAGE) withanti-human Fc antibodies. Lanes 1-5 were loaded as follows: 21 ofFc-L10-OSK1; 5 μl of Fc-L10-OSK1; 10 μl of Fc-L10-OSK1; 20 ng Human IgGstandard; molecular weight markers.

FIG. 48 shows a Western blot (from tris-glycine 4-20% SDS-PAGE) withanti-human Fc antibodies. Lanes 1-13 were loaded as follows: 20 ng HumanIgG standard; D1; C3; C2; B6; B5; B2; B1; A6; A5; A4; A3; A2 (5 μl ofclone-conditioned medium loaded in lanes 2-13).

FIG. 49A shows a spectral scan of 50 μl of the Fc-L10-OSK1 productdiluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 49B shows Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final Fc-L10-OSK1 product. Lane 1-12 are loaded asfollows: Novex Mark12 wide range protein standards, 0.5 μg productnon-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg productnon-reduced, Novex Mark12 wide range protein standards, 0.5 μg productreduced, blank, 2.0 μg product reduced, blank, and 10 μg productreduced.

FIG. 49C shows Size exclusion chromatography on 149 μg of the finalFc-L10-OSK1 product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm.

FIG. 49D shows MALDI mass spectral analysis of the final sample ofFc-L10-OsK1 analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses.

FIG. 50A shows a spectral scan of 50 μl of the Fc-L10-OsK1(K7S) productdiluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 50B shows Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final Fc-L10-OsK1(K7S) product. Lane 1-12 are loaded asfollows: Novex Mark12 wide range protein standards, 0.5 μg productnon-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg productnon-reduced, Novex Mark12 wide range protein standards, 0.5 μg productreduced, blank, 2.0 μg product reduced, blank, and 10 μg productreduced.

FIG. 50C shows size exclusion chromatography on 50 μg of the finalFc-L10-OsK1(K7S) product injected on to a Phenomenex BioSep SEC 3000column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/minobserving the absorbance at 280 nm.

FIG. 50D shows MALDI mass spectral analysis of a sample of the finalproduct Fc-L10-OsK1 (K7S) analyzed using a Voyager DE-RP time-of-flightmass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse).The positive ion/linear mode was used, with an accelerating voltage of25 kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses.

FIG. 51A shows a spectral scan of 50 μl of the Fc-L10-OsK1(E16K, K20D)product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard8453 spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 51B shows Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final Fc-L10-OsK1(E16K, K20D) product. Lane 1-12 areloaded as follows: Novex Mark12 wide range protein standards, 0.5 μgproduct non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μgproduct non-reduced, Novex Mark12 wide range protein standards, 0.5 μgproduct reduced, blank, 2.0 μg product reduced, blank, and 10 μg productreduced.

FIG. 51C shows size exclusion chromatography on 50 μg of the finalFc-L10-OsK1(E16K, K20D) product injected on to a Phenomenex BioSep SEC3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1ml/min observing the absorbance at 280 nm.

FIG. 51D shows MALDI mass spectral analysis of a sample of the finalproduct Fc-L10-OsK1 (E16K, K20D) analyzed using a Voyager DE-RPtime-of-flight mass spectrometer equipped with a nitrogen laser (337 nm,3 ns pulse). The positive ion/linear mode was used, with an acceleratingvoltage of 25 kV. Each spectrum was produced by accumulating data from˜200 laser shots. External mass calibration was accomplished usingpurified proteins of known molecular masses.

FIG. 52A shows a spectral scan of 50 μl of the Fc-L110-OsK1 (K7S, E16K,K20D) product diluted in 700 μl PBS (blanking buffer) using a HewlettPackard 8453 spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 52B shows Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final Fc-L10-OsK1(K7S, E16K, K20D) product. Lanes 1-12are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μgproduct non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μgproduct non-reduced, Novex Mark12 wide range protein standards, 0.5 μgproduct reduced, blank, 2.0 μg product reduced, blank, and 10 μg productreduced.

FIG. 52C shows size exclusion chromatography on 50 μg of the finalFc-L10-OsK1(K7S, E16K, K20D) product injected on to a Phenomenex BioSepSEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1ml/min observing the absorbance at 280 nm.

FIG. 52D shows MALDI mass spectral analysis of a sample of the finalproduct Fc-L10-OsK1(K7S, E16K, K20D) analyzed using a Voyager DE-RPtime-of-flight mass spectrometer equipped with a nitrogen laser (337 nm,3 ns pulse). The positive ion/linear mode was used, with an acceleratingvoltage of 25 kV. Each spectrum was produced by accumulating data from˜200 laser shots. External mass calibration was accomplished usingpurified proteins of known molecular masses.

FIG. 53 shows inhibition of the outward potassium current recorded fromHEK293 cell stably expressing human Kv1.3 channel by synthetic Osk1, a38-residue toxin peptide of the Asian scorpion Orthochirus scrobiculosusvenom. FIG. 53A shows a concentration dependent block of the outwardpotassium current recorded from HEK293 cell stably expressing humanKv1.3 channel by the synthetic Osk1 toxin peptide. FIG. 53B shows thetime course of the synthetic Osk1 toxin peptide block at variousconcentrations. The IC50 for the synthetic Osk1 toxin peptide wasestimated to be 39±12 pM (n=4 cells).

FIG. 54 shows that modification of the synthetic OSK1 toxin peptide byfusion to the Fc-fragment of an antibody (OSK1-peptibody) retained theinhibitory activity against the human Kv1.3 channel. FIG. 54A shows aconcentration dependent block of the outward potassium current recordedfrom HEK293 cells stably expressing human Kv1.3 channel by OSK1 linkedto a human IgG1 Fc-fragment with a linker chain length of 10 amino acidresidues (Fc-L10-OSK1). The fusion construct was stably expressed inChinese Hamster Ovarian (CHO) cells. FIG. 54B shows the time course ofthe Fc-L10-OSK1 block at various concentrations. The IC50 forFc-L10-OSK1 was estimated to be 198±35 pM (n=6 cells), approximately5-fold less potent than the synthetic OSK1 toxin peptide.

FIG. 55 shows that a single amino-acid residue substitution of theOSK1-peptibody retained the inhibitory activity against the human Kv1.3channel. FIG. 55A shows a concentration dependent block of the outwardpotassium current recorded from HEK293 cell stably expressing humanKv1.3 channel by OSK1-peptibody with a single amino acid substitution(lysine to serine at the 7^(th) position from N-terminal, [K7S]) andlinked to a human IgG1 Fc-fragment with a linker chain length of 10amino acid residues (Fc-L10-OSK1[K7S]). The fusion construct was stablyexpressed in Chinese Hamster Ovarian (CHO) cells. FIG. 55B shows thetime course of potassium current block by Fc-L10-OSK1[K7S] at variousconcentrations. The IC50 was estimated to be 372±71 pM (n=4 cells),approximately 10-fold less potent than the synthetic OSK1 toxin peptide.

FIG. 56 shows that a two amino-acid residue substitution of theOSK1-peptibody retained the inhibitory activity against the human Kv1.3channel. FIG. 56A shows a concentration dependent block of the outwardpotassium current recorded from HEK293 cell stably expressing humanKv1.3 channel by OSK1-peptibody with two amino acid substitutions(glutamic acid to lysine and lysine to aspartic acid at the 16^(th) and20^(th) position from N-terminal respectively, [E16KK20D]) and linked toa human IgG1 Fc-fragment with a linker chain length of 10 amino acidresidues (Fc-L10-OSK1[E16KK20D]). The fusion construct was stablyexpressed in Chinese Hamster Ovarian (CHO) cells. FIG. 56B shows thetime course of potassium current block by Fc-L10-OSK1[E16KK20D] atvarious concentrations. The IC50 was estimated to be 248±63 pM (n=3cells), approximately 6-fold less potent than the synthetic OSK1 toxinpeptide.

FIG. 57 shows that a triple amino-acid residue substitution of theOSK1-peptibody retained the inhibitory activity against the human Kv1.3channel, but the potency of inhibition was significantly reduced whencompared to the synthetic OSK1 toxin peptide. FIG. 57A shows aconcentration dependent block of the outward potassium current recordedfrom HEK293 cell stably expressing human Kv1.3 channel by OSK1-peptibodywith triple amino acid substitutions (lysine to serine, glutamic acid tolysine and lysine to aspartic acid at the 7^(th), 16^(th) and 20^(th)position from N-terminal respectively, [K7SE16KK20D]) and linked to ahuman IgG1 Fc-fragment with a linker chain length of 10 amino acidresidues (Fc-L10-OSK1[K7SE16KK20D]). The fusion construct was stablyexpressed in Chinese Hamster Ovarian (CHO) cells. FIG. 57B shows thetime course of potassium current block by Fc-L10-OSK1[K7SE16KK20D] atvarious concentrations. The IC50 was estimated to be 812±84 pM (n=3cells), approximately 21-fold less potent than the synthetic OSK1 toxinpeptide.

FIG. 58 shows Standard curves for ShK (FIG. 58A) and 20K PEG-ShK[1-35](FIG. 58B) containing linear regression equations for each Standard at agiven percentage of serum.

FIG. 59 shows the pharmacokinetic profile in rats of 20K PEG ShK[1-35]molecule after IV injection.

FIG. 60 shows Kv1.3 inhibitory activity in serum samples (5%) of ratsreceiving a single equal molar IV injection of Kv1.3 inhibitors ShKversus 20K PEG-ShK[1-35].

FIG. 61 illustrates an Adoptive Transfer EAE model experimental design(n=5 rats per treatment group). Dosing values in microgram per kilogram(mg/kg) are based on peptide content.

FIG. 62 shows that treatment with PEG-ShK ameliorated disease in rats inthe adoptive transfer EAE model. Clinical scoring: 0=No signs,0.5=distal limp tail, 1.0=limp tail, 2.0=mild paraparesis, ataxia,3.0=moderate paraparesis, 3.5=one hind leg paralysis, 4.0=complete hindleg paralysis, 5.0=complete hind leg paralysis and incontinence,5.5=tetraplegia, 6.0=moribund state or death. Rats reaching a score of5.5 to 6 died or were euthanized. Mean±sem values are shown. (n=5 ratsper treatment group.)

FIG. 63 shows that treatment with PEG-ShK prevented loss of body weightin the adoptive transfer EAE model. Rats were weighed on days −1, 4, 6,and 8 (for surviving rats). Mean±sem values are shown.

FIG. 64 shows that thapsigargin-induced IL-2 production in human wholeblood was suppressed by the Kv1.3 channel inhibitors ShK[1-35] andFc-L10-ShK[2-35]. The calcineurin inhibitor cyclosporine A also blockedthe response. The BKCa channel inhibitor iberiotoxin (IbTx) showed nosignificant activity. The response of whole blood from two separatedonors is shown in FIG. 64A and FIG. 64B.

FIG. 65 shows that thapsigargin-induced IFN-g production in human wholeblood was suppressed by the Kv1.3 channel inhibitors ShK[1-35] andFc-L10-ShK[2-35]. The calcineurin inhibitor cyclosporine A also blockedthe response. The BKCa channel inhibitor iberiotoxin (IbTx) showed nosignificant activity. The response of whole blood from two separatedonors is shown in FIG. 65A and FIG. 65B.

FIG. 66 shows that thapsigargin-induced upregulation of CD40L on T cellsin human whole blood was suppressed by the Kv1.3 channel inhibitorsShK[1-35] and Fc-L10-ShK[1-35] (Fc-ShK). The calcineurin inhibitorcyclosporine A (CsA) also blocked the response. FIG. 66A shows resultsof an experiment looking at the response of total CD4+ T cells. FIG. 66Bshows results of an experiment that looked at total CD4+ T cells, aswell as CD4+CD45+ and CD4+CD45-T cells. In FIG. 66B, the BKCa channelinhibitor iberiotoxin (IbTx) and the Kv1.1 channel inhibitordendrotoxin-K (DTX-K) showed no significant activity.

FIG. 67 shows that thapsigargin-induced upregulation of the IL-2R on Tcells in human whole blood was suppressed by the Kv1.3 channelinhibitors ShK[1-35] and Fc-L10-ShK[1-35] (Fc-ShK). The calcineurininhibitor cyclosporine A (CsA) also blocked the response. FIG. 67A showsresults of an experiment looking at the response of total CD4+ T cells.FIG. 67B shows results of an experiment that looked at total CD4+ Tcells, as well as CD4+CD45+ and CD4+CD45-T cells. In FIG. 67B, the BKCachannel inhibitor iberiotoxin (IbTx) and the Kv1.1 channel inhibitordendrotoxin-K (DTX-K) showed no significant activity.

FIG. 68 shows cation exchange chromatograms of PEG-peptide purificationon SP Sepharose HP columns for PEG-Shk purification (FIG. 68A) andPEG-OSK-1 purification (FIG. 68B).

FIG. 69 shows RP-HPLC chromatograms on final PEG-peptide pools todemonstrate purity of PEG-Shk purity >99% (FIG. 69A) and PEG-Osk1purity >97% (FIG. 69B).

FIG. 70 shows the amino acid sequence (SEQ ID NO: 976) of an exemplaryFcLoop-L2-OsK1-L2 having three linked domains: Fc N-terminal domain(amino acid residues 1-139); OsK1 (underlined amino acid residues142-179); and Fc C-terminal domain (amino acid residues 182-270).

FIG. 71 shows the amino acid sequence (SEQ ID NO: 977) of an exemplaryFcLoop-L2-ShK-L2 having three linked domains: Fc N-terminal domain(amino acid residues 1-139); ShK (underlined amino acid residues142-176); and Fc C-terminal domain (amino acid residues 179-267).

FIG. 72 shows the amino acid sequence (SEQ ID NO: 978) of an exemplaryFcLoop-L2-ShK-L4 having three linked domains: Fc N-terminal domain(amino acid residues 1-139); ShK (underlined amino acid residues142-176); and Fc C-terminal domain (amino acid residues 181-269).

FIG. 73 shows the amino acid sequence (SEQ ID NO: 979) of an exemplaryFcLoop-L4-OsK1-L2 having three linked domains: Fc N-terminal domain(amino acid residues 1-139); OsK1(underlined amino acid residues144-181); and Fc C-terminal domain (amino acid residues 184-272).

FIG. 74 shows that the 20K PEGylated ShK[1-35] provided potent blockadeof human Kv1.3 as determined by whole cell patch clamp electrophysiologyon HEK293/Kv1.3 cells. The data represents blockade of peak current.

FIG. 75 shows schematic structures of some other exemplary embodimentsof the composition of matter of the invention. “X²” and “X³” representtoxin peptides or linker-toxin peptide combinations (i.e.,-(L)_(f)-P-(L)_(g)-) as defined herein. As described herein but notshown in FIG. 75, an additional X¹ domain and one or more additional PEGmoieties are also encompassed in other embodiments. The specificembodiments shown here are as follows:

FIG. 75C, FIG. 75D, FIG. 75G and FIG. 75H: show a single chain moleculeand can also represent the DNA construct for the molecule.

FIG. 75A, FIG. 75B, FIG. 75E and FIG. 75F: show doubly disulfide-bondedFc dimers (in position F²); FIG. 75A and FIG. 75B show a dimer havingthe toxin peptide portion on both chains in position X³; FIG. 75E andFIG. 75F show a dimer having the toxin peptide portion on both chains Inposition X².

FIG. 76A shows a spectral scan of 50 μl of the ShK[2-35]-Fc productdiluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 76B shows Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final ShK[2-35]-Fc product. Lanes 1-12 were loaded asfollows: Novex Mark12 wide range protein standards, 0.5 μg productnon-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg productnon-reduced, Novex Mark12 wide range protein standards, 0.5 μg productreduced, blank, 2.0 μg product reduced, blank, and 10 μg productreduced.

FIG. 76C shows size exclusion chromatography on 70 μg of the finalShK[2-35]-Fc product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm.

FIG. 76D shows LC-MS analysis of the final ShK[2-35]-Fc sample using anAgilent 1100 HPCL running reverse phase chromatography, with the columneffluent directly coupled to an electrospray source of a Thermo FinniganLCQ ion trap mass spectrometer. Relevant spectra were summed anddeconvoluted to mass data with the Bioworks software package.

FIG. 77A shows a spectral scan of 20 μl of the met-ShK[1-35]-Fc productdiluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 77B shows Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final met-ShK[1-35]-Fc product. Lanes 1-12 were loadedas follows: Novex Mark12 wide range protein standards, 0.5 μg productnon-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg productnon-reduced, Novex Mark12 wide range protein standards, 0.5 μg productreduced, blank, 2.0 μg product reduced, blank, and 10 μg productreduced.

FIG. 77C shows size exclusion chromatography on 93 μg of the finalmet-ShK[1-35]-Fc product injected on to a Phenomenex BioSep SEC 3000column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/minobserving the absorbance at 280 nm.

FIG. 77D shows MALDI mass spectral analysis of the finalmet-ShK[1-35]-Fc sample analyzed using a Voyager DE-RP time-of-flightmass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse).The positive ion/linear mode was used, with an accelerating voltage of25 kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses.

FIG. 78 shows a spectral scan of 10 μl of the CH2-OSK1 fusion proteinproduct diluted in 150 μl water (blanking buffer) using a HewlettPackard 8453 spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 79 shows Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final CH2-OSK1 fusion protein product. Lane 1-7 wereloaded as follows: Novex Mark12 wide range protein standards, 0.5 μgproduct non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μgproduct non-reduced, and Novex Mark12 wide range protein standards.

FIG. 80 shows size exclusion chromatography on 50 μg of the finalCH2-OSK1 fusion protein product injected on to a Phenomenex BioSep SEC3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1ml/min observing the absorbance at 280 nm.

FIG. 81 shows liquid chromatography—mass spectral analysis of theCH2-OSK1 fusion protein sample using a Vydac C₄ column with part of theeffluent directed into a LCQ ion trap mass spectrometer. The massspectrum was deconvoluted using the Bioworks software provided by themass spectrometer manufacturer.

FIG. 82 shows cation exchange chromatogram of PEG-CH2-OSK1 reactionmixture. Vertical lines delineate fractions pooled to obtainmono-PEGylated CH2-OSK1.

FIG. 83 shows Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE of the final PEGylated CH2-OSK1 pool. Lane 1-2 were loaded asfollows: Novex Mark12 wide range protein standards, 2.0 μg productnon-reduced.

FIG. 84 shows whole cell patch clamp (WCPC) and PatchXpress (PX)electrophysiology comparing the activity of OSK1[Ala-12] (SEQ IDNo:1410) on human Kv1.3 and human Kv1.1 heterologously overexpressed onCHO and HEK293 cells, respectively. The table summarizes the calculatedIC₅₀ values and the plots show the individual traces of the impact ofvarious concentrations of analog on the relative Kv1.3 or Kv1.1 current(percent of control, POC).

FIG. 85 shows whole cell patch clamp electrophysiology comparing theactivity of OSK1[Ala-29] (SEQ ID No:1424) on human Kv1.3 and human Kv1.1heterologously overexpressed on CHO and HEK293 cells, respectively.Concentration response curves of OSK1[Ala-29] on CHO/Kv1.3 (circle,square and diamond, IC₅₀=0.033 nM, n=3) and on HEK/Kv1.1 (filledtriangle, IC₅₀=2.7 nM, n=1).

FIG. 86 shows a dose-response curve for OSK1[Ala-29] (SEQ ID No:1424)against human Kv1.3 (CHO) (panel A) and human Kv1.1 (HEK293) (panel B)as determined by high-throughput 384-well planar patch clampelectrophysiology using the IonWorks Quattro system.

FIG. 87A-B show Western blots of Tris-glycine 4-20% SDS-PAGE (FIG. 87Awith longer exposure time and FIG. 87B with shorter exposure time) of amonovalent dimeric Fc-L-ShK(2-35) molecule product expressed by andreleased into the conditioned media from mammalian cells transientlytransfected with pTT5-Fc-Fc-L10-Shk(2-35), which was sampled after theindicated number of days. Lanes 3-8 were loaded with 20 μL ofconditioned medium per lane. The immunoblot was probed with anti-humanIgG-Fc-HRP (Pierce). The lanes were loaded as follows: 1) MW Markers; 2)purified Fc-L10-ShK(2-35), 100 ng; 3) 293-6E-HD (5-day); 4) 293-6E-HD(6-day); 5) 293-6E-PEI (5-day); 6) 293-6E-PEI (6-day); 7) CHO-S (5-day);8) CHO-S (6-day). Four bands were expected in the reduced gel:Linker-Fc-Shk(2-35) (one cut at 3′ furin site; predicted MW: 33.4 kDa);Fc-ShK(2-35) (both furin sites cut; predicted MW: 30.4 kDa); Fc-linker(one cut at 5′ furin site; predicted MW: 29.1 kDa); Fc (both furin sitescut; predicted MW: 25.8 kDa). Further mass spec or amino acid sequenceanalysis of the individual bands is needed to identify these bands andtheir relative ratios.

FIG. 88 shows a western blot of serum samples from a pharmacokineticstudy on monovalent dimeric Fc-ShK(1-35) in SD rats. Various times(0.083-168 hours) after a single 1 mg/kg intravenous injection ofmonovalent dimeric Fc-L10-ShK(1-35) (see, Example 2), blood was drawn,and serum was collected. A Costar EIA/RIA 96 well plate was coated with2 μg/ml polyclonal goat anti-human Fc antibody overnight at 4° C.Capture antibody was removed and the plate was washed with PBST and thenblocked with Blotto. After the plate was washed, serum samples dilutedin PBST/0.1% BSA were added. Binding was allowed to occur at roomtemperature for several hours, and then the plate was again washed.Samples were eluted from the plate with reducing Laemmle buffer, heated,then run on SDS-Page gels. Run in an adjacent lane (“5 ng Control”) ofthe gel as a standard was 5 ng of the purified monovalent dimericFc-L10-ShK(1-35) fusion protein used in the pharmacokinetic study.Proteins were transferred to PVDF membranes by western blot. Membraneswere blocked with Blotto followed by incubation with goat anti-HumanFc-HRP conjugated antibody. After the membranes were washed, signal wasdetected via chemiluminescence using a CCD camera.

FIG. 89 shows the NMR solution structure of OSK1 and sites identified byanaloging to be important for Kv1.3 activity and selectivity. Spacefilling structures are shown in FIGS. 89A, 89B and 89D. The colorrendering in FIG. 89A depicts amino acid charge. In FIG. 89B, severalkey OSK1 amino acid residues found to be important for Kv1.3 activity(Tables 37-40) are lightly shaded and labeled Phe25, Gly26, Lys27, Met29and Asn30. In FIG. 89D residues Ser11, Met29 and His34 are labeled. Someanalogues of these residues were found to result in improved Kv1.3selectivity over Kv1.1 (Tables 41). FIG. 89C shows the three betastrands and single alpha helix of OSK1. The amino acid sequence ofnative OSK1 (SEQ ID No: 25) is shown in FIG. 89E, with residues formingthe molecules beta strands (β1, β2, β3) and alpha helix (al) underlined.The OSK1 structures shown were derived from PDB:1SCO, and were renderedusing Cn3D vers4.1.

FIG. 90A-D illustrates that toxin peptide inhibitors of Kv1.3 providepotent blockade of the whole blood inflammatory response. The activityof the calcineurin inhibitor cyclosporin A (FIG. 90A) and Kv1.3 peptideinhibitors ShK-Ala22 (FIG. 90B; SEQ ID No: 123), OSK1-Ala29 (FIG. 90C;SEQ ID No: 1424) and OSK1-Ala12 (FIG. 90D; SEQ ID No: 1410) werecompared in the whole blood assay of inflammation (Example 46) using thesame donor blood sample. The potency (IC50) of each molecule is shown,where for each panel the left curve is the impact on IL-2 production andthe right curve is the impact on IFNγ production.

FIG. 91A-B shows an immunoblot analysis of expression of monovalentdimeric IgG1-Fc-L-ShK(2-35) from non-reduced SDS-PAGE. FIG. 91A showsdetection of human Fc expression with goat anti-human IgG (H+L)-HRP.FIG. 91B shows detection of ShK(2-35) expression with a goat anti-mouseIgG (H+L)-HRP that cross reacts with human IgG. Lane 1: purifiedFc-L10-Shk(2-35); Lane 2: conditioned medium from 293EBNA cellstransiently transfected withpTT5-huIgG1+pTT5-hKappa+pCMVi-Fc-L10-ShK(2-35); Lane 3: conditionedmedia from 293EBNA cells transiently transfected withpTT5-huIgG2+pTT5-hKappa+pCMVi-Fc-L10-Shk(2-35); Lane 4: conditionedmedia from 293EBNA cells transiently transfected with pTT14 vectoralone. The two arrows point to the full length huIgG (mol. wt.˜150 kDa)and monovalent dimeric huIgG-FcShK(2-35) (mol. wt.˜100 kDa); theabundant 60-kDa band is the bivalent dimeric Fc-ShK(2-35).

FIG. 92A-C shows schematic representations of an embodiment of amonovalent “hemibody”-toxin peptide fusion protein construct; the singletoxin peptide is represented by an oval. FIG. 92A, which can alsorepresent the DNA construct for the fusion protein, represents animmunoglobulin light chain (LC, open rectangle), an immunoglobulin heavychain (HC, longer cross-hatched rectangle), and an immunoglobulin Fcdomain (Fc, shorter cross-hatched rectangle), each separated by anintervening peptidyl linker sequence (thick lines) comprising at leastone protease cleavage site (arrows), e.g., a furin cleavage site. FIG.92 illustrates the association of the recombinantly expressed LC, HC,and Fc-toxin peptide components connected by the peptidyl linkersequences (thick lines) and, in FIG. 92C, the final monovalent chimericimmunoglobulin (LC+HC)-Fc (i.e., “hemibody”)-toxin peptide fusionprotein after cleavage (intracellularly or extracellularly) at theprotease cleavage sites, to release the linkers, and formation ofdisulfide bridges between the light and heavy chains and between theheavy chain and the Fc components (shown as thin horizontal linesbetween the LC, HC, and Fc components in FIG. 92C).

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Definition of Terms

The terms used throughout this specification are defined as follows,unless otherwise limited in specific instances. As used in thespecification and the appended claims, the singular forms “a”, “an”, and“the” include plural referents unless the context clearly dictatesotherwise.

“Polypeptide” and “protein” are used interchangeably herein and includea molecular chain of two or more amino acids linked through peptidebonds. The terms do not refer to a specific length of the product. Thus,“peptides,” and “oligopeptides,” are included within the definition ofpolypeptide. The terms include post-translational modifications of thepolypeptide, for example, glycosylations, acetylations, phosphorylationsand the like. In addition, protein fragments, analogs, mutated orvariant proteins, fusion proteins and the like are included within themeaning of polypeptide. The terms also include molecules in which one ormore amino acid analogs or non-canonical or unnatural amino acids areincluded as can be synthesized, or expressed recombinantly using knownprotein engineering techniques. In addition, inventive fusion proteinscan be derivatized as described herein by well-known organic chemistrytechniques.

The term “fusion protein” indicates that the protein includespolypeptide components derived from more than one parental protein orpolypeptide. Typically, a fusion protein is expressed from a fusion genein which a nucleotide sequence encoding a polypeptide sequence from oneprotein is appended in frame with, and optionally separated by a linkerfrom, a nucleotide sequence encoding a polypeptide sequence from adifferent protein. The fusion gene can then be expressed by arecombinant host cell as a single protein.

A “domain” of a protein is any portion of the entire protein, up to andincluding the complete protein, but typically comprising less than thecomplete protein. A domain can, but need not, fold independently of therest of the protein chain and/or be correlated with a particularbiological, biochemical, or structural function or location (e.g., aligand binding domain, or a cytosolic, transmembrane or extracellulardomain).

A “secreted” protein refers to those proteins capable of being directedto the ER, secretory vesicles, or the extracellular space as a result ofa secretory signal peptide sequence, as well as those proteins releasedinto the extracellular space without necessarily containing a signalsequence. If the secreted protein is released into the extracellularspace, the secreted protein can undergo extracellular processing toproduce a “mature” protein. Release into the extracellular space canoccur by many mechanisms, including exocytosis and proteolytic cleavage.

The term “signal peptide” refers to a relatively short (3-60 amino acidresidues long) peptide chain that directs the post-translationaltransport of a protein, e.g., its export to the extracellular space.Thus, secretory signal peptides are encompassed by “signal peptide”.Signal peptides may also be called targeting signals, signal sequences,transit peptides, or localization signals.

The term “recombinant” indicates that the material (e.g., a nucleic acidor a polypeptide) has been artificially or synthetically (i.e.,non-naturally) altered by human intervention. The alteration can beperformed on the material within, or removed from, its naturalenvironment or state. For example, a “recombinant nucleic acid” is onethat is made by recombining nucleic acids, e.g., during cloning, DNAshuffling or other well known molecular biological procedures. A“recombinant DNA molecule,” is comprised of segments of DNA joinedtogether by means of such molecular biological techniques. The term“recombinant protein” or “recombinant polypeptide” as used herein refersto a protein molecule which is expressed using a recombinant DNAmolecule. A “recombinant host cell” is a cell that contains and/orexpresses a recombinant nucleic acid.

A “polynucleotide sequence” or “nucleotide sequence” or “nucleic acidsequence,” as used interchangeably herein, is a polymer of nucleotides,including an oligonucleotide, a DNA, and RNA, a nucleic acid, or acharacter string representing a nucleotide polymer, depending oncontext. From any specified polynucleotide sequence, either the givennucleic acid or the complementary polynucleotide sequence can bedetermined. Included are DNA or RNA of genomic or synthetic origin whichmay be single- or double-stranded, and represent the sense or antisensestrand.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of ribonucleotidesalong the mRNA chain, and also determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for the RNAsequence and for the amino acid sequence.

“Expression of a gene” or “expression of a nucleic acid” meanstranscription of DNA into RNA (optionally including modification of theRNA, e.g., splicing), translation of RNA into a polypeptide (possiblyincluding subsequent post-translational modification of thepolypeptide), or both transcription and translation, as indicated by thecontext.

The term “gene” is used broadly to refer to any nucleic acid associatedwith a biological function. Genes typically include coding sequencesand/or the regulatory sequences required for expression of such codingsequences. The term “gene” applies to a specific genomic or recombinantsequence, as well as to a cDNA or mRNA encoded by that sequence. A“fusion gene” contains a coding region that encodes a fusion protein.Genes also include non-expressed nucleic acid segments that, forexample, form recognition sequences for other proteins. Non-expressedregulatory sequences including transcriptional control elements to whichregulatory proteins, such as transcription factors, bind, resulting intranscription of adjacent or nearby sequences.

As used herein the term “coding region” when used in reference to astructural gene refers to the nucleotide sequences which encode theamino acids found in the nascent polypeptide as a result of translationof an mRNA molecule. The coding region is bounded, in eukaryotes, on the5′ side by the nucleotide triplet “ATG” which encodes the initiatormethionine and on the 3′ side by one of the three triplets which specifystop codons (i.e., TAA, TAG, TGA).

Transcriptional control signals in eukaryotes comprise “promoter” and“enhancer” elements. Promoters and enhancers consist of short arrays ofDNA sequences that interact specifically with cellular proteins involvedin transcription (Maniatis, et al., Science 236:1237 (1987)). Promoterand enhancer elements have been isolated from a variety of eukaryoticsources including genes in yeast, insect and mammalian cells and viruses(analogous control elements, i.e., promoters, are also found inprokaryotes). The selection of a particular promoter and enhancerdepends on what cell type is to be used to express the protein ofinterest. Some eukaryotic promoters and enhancers have a broad hostrange while others are functional in a limited subset of cell types (forreview see Voss, et al., Trends Biochem. Sci., 11:287 (1986) andManiatis, et al., Science 236:1237 (1987)).

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host cell. Nucleic acid sequencesnecessary for expression in prokaryotes include a promoter, optionallyan operator sequence, a ribosome binding site and possibly othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals. A secretory signal peptidesequence can also, optionally, be encoded by the expression vector,operably linked to the coding sequence for the inventive recombinantfusion protein, so that the expressed fusion protein can be secreted bythe recombinant host cell, for more facile isolation of the fusionprotein from the cell, if desired. Such techniques are well known in theart. (E.g., Goodey, Andrew R.; et al., Peptide and DNA sequences, U.S.Pat. No. 5,302,697; Weiner et al., Compositions and methods for proteinsecretion, U.S. Pat. Nos. 6,022,952 and 6,335,178; Uemura et al.,Protein expression vector and utilization thereof, U.S. Pat. No.7,029,909; Ruben et al., 27 human secreted proteins, US 2003/0104400A1).

The terms “in operable combination”, “in operable order” and “operablylinked” as used herein refer to the linkage of nucleic acid sequences insuch a manner or orientation that a nucleic acid molecule capable ofdirecting the transcription of a given gene and/or the synthesis of adesired protein molecule is produced. The term also refers to thelinkage of amino acid sequences in such a manner so that a functionalprotein is produced and/or transported.

Recombinant DNA- and/or RNA-mediated protein expression techniques, orany other methods of preparing peptides or, are applicable to the makingof the inventive recombinant fusion proteins. For example, the peptidescan be made in transformed host cells. Briefly, a recombinant DNAmolecule, or construct, coding for the peptide is prepared. Methods ofpreparing such DNA molecules are well known in the art. For instance,sequences encoding the peptides can be excised from DNA using suitablerestriction enzymes. Any of a large number of available and well-knownhost cells may be used in the practice of this invention. The selectionof a particular host is dependent upon a number of factors recognized bythe art. These include, for example, compatibility with the chosenexpression vector, toxicity of the peptides encoded by the DNA molecule,rate of transformation, ease of recovery of the peptides, expressioncharacteristics, bio-safety and costs. A balance of these factors mustbe struck with the understanding that not all hosts may be equallyeffective for the expression of a particular DNA sequence. Within thesegeneral guidelines, useful microbial host cells in culture includebacteria (such as Escherichia coli sp.), yeast (such as Saccharomycessp.) and other fungal cells, insect cells, plant cells, mammalian(including human) cells, e.g., CHO cells and HEK293 cells. Modificationscan be made at the DNA level, as well. The peptide-encoding DNA sequencemay be changed to codons more compatible with the chosen host cell. ForE. coli, optimized codons are known in the art. Codons can besubstituted to eliminate restriction sites or to include silentrestriction sites, which may aid in processing of the DNA in theselected host cell. Next, the transformed host is cultured and purified.Host cells may be cultured under conventional fermentation conditions sothat the desired compounds are expressed. Such fermentation conditionsare well known in the art.

The term “half-life extending moiety” (i.e., F¹ or F² in Formula I)refers to a pharmaceutically acceptable moiety, domain, or “vehicle”covalently linked (“conjugated”) to the toxin peptide directly or via alinker, that prevents or mitigates in vivo proteolytic degradation orother activity-diminishing chemical modification of the toxin peptide,increases half-life or other pharmacokinetic properties such as but notlimited to increasing the rate of absorption, reduces toxicity, improvessolubility, increases biological activity and/or target selectivity ofthe toxin peptide with respect to a target ion channel of interest,increases manufacturability, and/or reduces immunogenicity of the toxinpeptide, compared to an unconjugated form of the toxin peptide.

By “PEGylated peptide” is meant a peptide or protein having apolyethylene glycol (PEG) moiety covalently bound to an amino acidresidue of the peptide itself or to a peptidyl or non-peptidyl linker(including but not limited to aromatic or aryl linkers) that iscovalently bound to a residue of the peptide.

By “polyethylene glycol” or “PEG” is meant a polyalkylene glycolcompound or a derivative thereof, with or without coupling agents orderivatization with coupling or activating moieties (e.g., withaldehyde, hydroxysuccinimidyl, hydrazide, thiol, triflate, tresylate,azirdine, oxirane, orthopyridyl disulphide, vinylsulfone, iodoacetamideor a maleimide moiety). In accordance with the present invention, usefulPEG includes substantially linear, straight chain PEG, branched PEG, ordendritic PEG. (See, e.g., Merrill, U.S. Pat. No. 5,171,264; Harris etal., Multiarmed, monofunctional, polymer for coupling to molecules andsurfaces, U.S. Pat. No. 5,932,462; Shen, N-maleimidyl polymerderivatives, U.S. Pat. No. 6,602,498).

The term “peptibody” refers to molecules of Formula I in which F¹ and/orF² is an immunoglobulin Fc domain or a portion thereof, such as a CH2domain of an Fc, or in which the toxin peptide is inserted into a humanIgG1 Fc domain loop, such that F¹ and F² are each a portion of an Fcdomain with a toxin peptide inserted between them (See, e.g., FIGS.70-73 and Example 49 herein). Peptibodies of the present invention canalso be PEGylated as described further herein, at either an Fc domain orportion thereof, or at the toxin peptide(s) portion of the inventivecomposition, or both.

The term “native Fc” refers to molecule or sequence comprising thesequence of a non-antigen-binding fragment resulting from digestion ofwhole antibody, whether in monomeric or multimeric form. The originalimmunoglobulin source of the native Fc is preferably of human origin andcan be any of the immunoglobulins, although IgG1 or IgG2 are preferred.Native Fc's are made up of monomeric polypeptides that can be linkedinto dimeric or multimeric forms by covalent (i.e., disulfide bonds) andnon-covalent association. The number of intermolecular disulfide bondsbetween monomeric subunits of native Fc molecules ranges from 1 to 4depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2,IgG3, IgG4, IgA1, IgGA2). One example of a native Fc is adisulfide-bonded dimer resulting from papain digestion of an IgG (seeEllison et al. (1982), Nucleic Acids Res. 10: 4071-9). The term “nativeFc” as used herein is generic to the monomeric, dimeric, and multimericforms.

The term “Fc variant” refers to a molecule or sequence that is modifiedfrom a native Fc but still comprises a binding site for the salvagereceptor, FcRn. Several published patent documents describe exemplary Fcvariants, as well as interaction with the salvage receptor. SeeInternational Applications WO 97/34 631 (published 25 Sep. 1997; WO96/32 478, corresponding to U.S. Pat. No. 6,096,891, issued Aug. 1,2000, hereby incorporated by reference in its entirety; and WO 04/110472. Thus, the term “Fc variant” includes a molecule or sequence that ishumanized from a non-human native Fc. Furthermore, a native Fc comprisessites that can be removed because they provide structural features orbiological activity that are not required for the fusion molecules ofthe present invention. Thus, the term “Fc variant” includes a moleculeor sequence that lacks one or more native Fc sites or residues thataffect or are involved in (1) disulfide bond formation, (2)incompatibility with a selected host cell (3) N-terminal heterogeneityupon expression in a selected host cell, (4) glycosylation, (5)interaction with complement, (6) binding to an Fc receptor other than asalvage receptor, or (7) antibody-dependent cellular cytotoxicity(ADCC). Fc variants are described in further detail hereinafter.

The term “Fc domain” encompasses native Fc and Fc variant molecules andsequences as defined above. As with Fc variants and native Fc's, theterm “Fc domain” includes molecules in monomeric or multimeric form,whether digested from whole antibody or produced by other means.

The term “multimer” as applied to Fc domains or molecules comprising Fcdomains refers to molecules having two or more polypeptide chainsassociated covalently, noncovalently, or by both covalent andnon-covalent interactions. IgG molecules typically form dimers; IgM,pentamers; IgD, dimers; and IgA, monomers, dimers, trimers, ortetramers. One skilled in the art can form multimers by exploiting thesequence and resulting activity of the native Ig source of the Fc or byderivatizing (as defined below) such a native Fc.

The term “dimer” as applied to Fc domains or molecules comprising Fcdomains refers to molecules having two polypeptide chains associatedcovalently or non-covalently. Thus, exemplary dimers within the scope ofthis invention are as shown in FIG. 2. A “monovalent dimeric” Fc-toxinpeptide fusion, or “monovalent dimer”, is a Fc-toxin peptide fusion thatincludes a toxin peptide conjugated with only one of the dimerized Fcdomains (e.g., as represented schematically in FIG. 2B). A “bivalentdimeric” Fc-toxin peptide fusion, or “bivalent dimer”, is a Fc-toxinpeptide fusion having both of the dimerized Fc domains each conjugatedseparately with a toxin peptide (e.g., as represented schematically inFIG. 2C).

The terms “derivatizing” and “derivative” or “derivatized” compriseprocesses and resulting compounds respectively in which (1) the compoundhas a cyclic portion; for example, cross-linking between cysteinylresidues within the compound; (2) the compound is cross-linked or has across-linking site; for example, the compound has a cysteinyl residueand thus forms cross-linked dimers in culture or in vivo; (3) one ormore peptidyl linkage is replaced by a non-peptidyl linkage; (4) theN-terminus is replaced by —NRR¹, NRC(O)R¹, —NRC(O)OR¹, —NRS(O)₂R¹,—NHC(O)NHR, a succinimide group, or substituted or unsubstitutedbenzyloxycarbonyl-NH—, wherein R and R¹ and the ring substituents are asdefined hereinafter; (5) the C-terminus is replaced by —C(O)R² or —NR³R⁴wherein R², R³ and R⁴ are as defined hereinafter; and (6) compounds inwhich individual amino acid moieties are modified through treatment withagents capable of reacting with selected side chains or terminalresidues. Derivatives are further described hereinafter.

The term “peptide” refers to molecules of 2 to about 80 amino acidresidues, with molecules of about 10 to about 60 amino acid residuespreferred and those of about 30 to about 50 amino acid residues mostpreferred. Exemplary peptides can be randomly generated by any knownmethod, carried in a peptide library (e.g., a phage display library), orderived by digestion of proteins. In any peptide portion of theinventive compositions, for example a toxin peptide or a peptide linkermoiety described herein, additional amino acids can be included oneither or both of the N- or C-termini of the given sequence. Of course,these additional amino acid residues should not significantly interferewith the functional activity of the composition.

“Toxin peptides” include peptides having the same amino acid sequence ofa naturally occurring pharmacologically active peptide that can beisolated from a venom, and also include modified peptide analogs(spelling used interchangeably with “analogues”) of such naturallyoccurring molecules.

The term “peptide analog” refers to a peptide having a sequence thatdiffers from a peptide sequence existing in nature by at least one aminoacid residue substitution, internal addition, or internal deletion of atleast one amino acid, and/or amino- or carboxy-terminal end truncations,or additions). An “internal deletion” refers to absence of an amino acidfrom a sequence existing in nature at a position other than the N- orC-terminus. Likewise, an “internal addition” refers to presence of anamino acid in a sequence existing in nature at a position other than theN- or C-terminus. “Toxin peptide analogs”, such as, but not limited to,an OSK1 peptide analog, ShK peptide analog, or ChTx peptide analog,contain modifications of a native toxin peptide sequence of interest(e.g., amino acid residue substitutions, internal additions orinsertions, internal deletions, and/or amino- or carboxy-terminal endtruncations, or additions as previously described above) relative to anative toxin peptide sequence of interest, which is in the case of OSK1:GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK (SEQ ID NO:25).

Examples of toxin peptides useful in practicing the present inventionare listed in Tables 1-32. The toxin peptide (“P”, or equivalently shownas “P¹” in FIG. 2) comprises at least two intrapeptide disulfide bonds,as shown, for example, in FIG. 9. Accordingly, this invention concernsmolecules comprising:

-   -   a) C¹-C³ and C²-C⁴ disulfide bonding in which C¹, C², C³, and C⁴        represent the order in which cysteine residues appear in the        primary sequence of the toxin peptide stated conventionally with        the N-terminus of the peptide on the left, with the first and        third cysteines in the amino acid sequence forming a disulfide        bond, and the second and fourth cysteines forming a disulfide        bond. Examples of toxin peptides with such a C¹-C³, C²-C⁴        disulfide bonding pattern include, but are not limited to,        apamin peptides, α-conopeptides, PnIA peptides, PnIB peptides,        and MII peptides, and analogs of any of the foregoing.    -   b) C¹-C⁶, C²-C⁴ and C³-C⁵ disulfide bonding in which, as        described above, C¹, C², C³, C⁴, C⁵ and C⁶ represent the order        of cysteine residues appearing in the primary sequence of the        toxin peptide stated conventionally with the N-terminus of the        peptide(s) on the left, with the first and sixth cysteines in        the amino acid sequence forming a disulfide bond, the second and        fourth cysteines forming a disulfide bond, and the third and        fifth cysteines forming a disulfide bond. Examples of toxin        peptides with such a C¹-C⁶, C²-C⁴, C³-C⁵ disulfide bonding        pattern include, but are not limited to, ShK, BgK, HmK, AeKS,        AsK, and DTX1, and analogs of any of the foregoing.    -   c) C¹-C⁴, C²-C⁵ and C³-C⁶ disulfide bonding in which, as        described above, C¹, C², C³, C⁴, C⁵ and C⁶ represent the order        of cysteine residues appearing in the primary sequence of the        toxin peptide stated conventionally with the N-terminus of the        peptide(s) on the left, with the first and fourth cysteines in        the amino acid sequence forming a disulfide bond, the second and        fifth cysteines forming a disulfide bond, and the third and        sixth cysteines forming a disulfide bond. Examples of toxin        peptides with such a C¹-C⁴, C²-C⁵, C³-C⁶ disulfide bonding        pattern include, but are not limited to, ChTx, MgTx, OSK1, KTX1,        AgTx2, Pi2, Pi3, NTX, HgTx1, BeKM1, BmKTX, P01, BmKK6, Tc32,        Tc1, BmTx1, BmTX3, IbTx, P05, ScyTx, TsK, HaTx1, ProTX1, PaTX2,        Ptu1, ωGVIA, ωMVIIA, and SmIIIa, and analogs of any of the        foregoing.    -   d) C¹-C⁵, C²-C⁶, C³-C⁷, and C⁴-C⁸ disulfide bonding in which C¹,        C², C³, C⁴, C⁵, C⁶, C⁷ and C⁸ represent the order of cysteine        residues appearing in the primary sequence of the toxin peptide        stated conventionally with the N-terminus of the peptide(s) on        the left, with the first and fifth cysteines in the amino acid        sequence forming a disulfide bond, the second and sixth        cysteines forming a disulfide bond, the third and seventh        cysteines forming a disulfide bond, and the fourth and eighth        cysteines forming a disulfide bond. Examples of toxin peptides        with such a C¹-C⁵, C²-C⁶, C³-C⁷, C⁴-C⁸ disulfide bonding pattern        include, but are not limited to, Anuoroctoxin (AnTx), Pi1,        HsTx1, MTX (P12A, P20A), and Pi4 peptides, and analogs of any of        the foregoing.    -   e) C¹-C⁴, C²-C⁶, C³-C⁷, and C⁵-C⁸ disulfide bonding in which C⁷,        C², C³, C⁴, C⁵, C⁶, C⁷ and C⁸ represent the order of cysteine        residues appearing in the primary sequence of the toxin peptide        stated conventionally with the N-terminus of the peptide(s) on        the left, with the first and fourth cysteines in the amino acid        sequence forming a disulfide bond, the second and sixth        cysteines forming a disulfide bond, the third and seventh        cysteines forming a disulfide bond, and the fifth and eighth        cysteines forming a disulfide bond. Examples of toxin peptides        with such a C¹-C⁴, C²-C⁶, C³-C⁷, C⁵-C⁸ disulfide bonding pattern        include, but are not limited to, Chlorotoxin, Bm-12b, and, and        analogs of either.    -   f) C¹-C⁵, C²-C⁶, C³-C⁴, and C⁷-C⁸ disulfide bonding in which C¹,        C², C³, C⁴, C⁵, C⁶, C⁷ and C⁸ represent the order of cysteine        residues appearing in the primary sequence of the toxin peptide        stated conventionally with the N-terminus of the peptide(s) on        the left, with the first and fifth cysteines in the amino acid        sequence forming a disulfide bond, the second and sixth        cysteines forming a disulfide bond, the third and fourth        cysteines forming a disulfide bond, and the seventh and eighth        cysteines forming a disulfide bond. Examples of toxin peptides        with such a C¹-C⁵, C²-C⁶, C³-C⁴, C⁷-C⁸ disulfide bonding pattern        include, but are not limited to, Maurotoxin peptides and analogs        thereof.

The term “randomized” as used to refer to peptide sequences refers tofully random sequences (e.g., selected by phage display methods) andsequences in which one or more residues of a naturally occurringmolecule is replaced by an amino acid residue not appearing in thatposition in the naturally occurring molecule. Exemplary methods foridentifying peptide sequences include phage display, E. coli display,ribosome display, yeast-based screening, RNA-peptide screening, chemicalscreening, rational design, protein structural analysis, and the like.

The term “pharmacologically active” means that a substance so describedis determined to have activity that affects a medical parameter (e.g.,blood pressure, blood cell count, cholesterol level) or disease state(e.g., cancer, autoimmune disorders). Thus, pharmacologically activepeptides comprise agonistic or mimetic and antagonistic peptides asdefined below.

The terms “-mimetic peptide” and “-agonist peptide” refer to a peptidehaving biological activity comparable to a naturally occurring toxinpeptide molecule, e.g., naturally occurring ShK toxin peptide. Theseterms further include peptides that indirectly mimic the activity of anaturally occurring toxin peptide molecule, such as by potentiating theeffects of the naturally occurring molecule.

The term “antagonist peptide” or “inhibitor peptide” refers to a peptidethat blocks or in some way interferes with the biological activity of areceptor of interest, or has biological activity comparable to a knownantagonist or inhibitor of a receptor of interest (such as, but notlimited to, an ion channel).

The term “acidic residue” refers to amino acid residues in D- or L-formhaving sidechains comprising acidic groups. Exemplary acidic residuesinclude D and E.

The term “amide residue” refers to amino acids in D- or L-form havingsidechains comprising amide derivatives of acidic groups. Exemplaryresidues include N and Q.

The term “aromatic residue” refers to amino acid residues in D- orL-form having sidechains comprising aromatic groups. Exemplary aromaticresidues include F, Y, and W.

The term “basic residue” refers to amino acid residues in D- or L-formhaving sidechains comprising basic groups. Exemplary basic residuesinclude H, K, R, N-methyl-arginine, ω-aminoarginine, ω-methyl-arginine,1-methyl-histidine, 3-methyl-histidine, and homoarginine (hR) residues.

The term “hydrophilic residue” refers to amino acid residues in D- orL-form having sidechains comprising polar groups. Exemplary hydrophilicresidues include C, S, T, N, Q, D, E, K, and citrulline (Cit) residues.

The term “nonfunctional residue” refers to amino acid residues in D- orL-form having sidechains that lack acidic, basic, or aromatic groups.Exemplary nonfunctional amino acid residues include M, G, A, V, I, L andnorleucine (Nle).

The term “neutral polar residue” refers to amino acid residues in D- orL-form having sidechains that lack basic, acidic, or polar groups.Exemplary neutral polar amino acid residues include A, V, L, I, P, W, M,and F.

The term “polar hydrophobic residue” refers to amino acid residues in D-or L-form having sidechains comprising polar groups. Exemplary polarhydrophobic amino acid residues include T, G, S, Y, C, Q, and N.

The term “hydrophobic residue” refers to amino acid residues in D- orL-form having sidechains that lack basic or acidic groups. Exemplaryhydrophobic amino acid residues include A, V, L, I, P, W, M, F, T, G, S,Y, C, Q, and N.

In some useful embodiments of the compositions of the invention, theamino acid sequence of the toxin peptide is modified in one or more waysrelative to a native toxin peptide sequence of interest, such as, butnot limited to, a native ShK or OSK1 sequence, their peptide analogs, orany other toxin peptides having amino acid sequences as set for in anyof Tables 1-32. The one or more useful modifications can include aminoacid additions or insertions, amino acid deletions, peptide truncations,amino acid substitutions, and/or chemical derivatization of amino acidresidues, accomplished by known chemical techniques. Such modificationscan be, for example, for the purpose of enhanced potency, selectivity,and/or proteolytic stability, or the like. Those skilled in the art areaware of techniques for designing peptide analogs with such enhancedproperties, such as alanine scanning, rational design based on alignmentmediated mutagenesis using known toxin peptide sequences and/ormolecular modeling. For example, ShK analogs can be designed to removeprotease cleavage sites (e.g., trypsin cleavage sites at K or R residuesand/or chymotrypsin cleavage sites at F, Y, or W residues) in a ShKpeptide- or ShK analog-containing composition of the invention, basedpartially on alignment mediated mutagenesis using HmK (see, e.g., FIG.6) and molecular modeling. (See, e.g., Kalman et al., ShK-Dap22, apotent Kv1.3-specific immunosuppressive polypeptide, J. Biol. Chem.273(49):32697-707 (1998); Kem et al., U.S. Pat. No. 6,077,680; Mouhat etal., OsK1 derivatives, WO 2006/002850 A2)).

The term “protease” is synonymous with “peptidase”. Proteases comprisetwo groups of enzymes: the endopeptidases which cleave peptide bonds atpoints within the protein, and the exopeptidases, which remove one ormore amino acids from either N- or C-terminus respectively. The term“proteinase” is also used as a synonym for endopeptidase. The fourmechanistic classes of proteinases are: serine proteinases, cysteineproteinases, aspartic proteinases, and metallo-proteinases. In additionto these four mechanistic classes, there is a section of the enzymenomenclature which is allocated for proteases of unidentified catalyticmechanism. This indicates that the catalytic mechanism has not beenidentified.

Cleavage subsite nomenclature is commonly adopted from a scheme createdby Schechter and Berger (Schechter I. & Berger A., On the size of theactive site in proteases. I. Papain, Biochemical and BiophysicalResearch Communication, 27:157 (1967); Schechter I. & Berger A., On theactive site of proteases. 3. Mapping the active site of papain; specificinhibitor peptides of papain, Biochemical and Biophysical ResearchCommunication, 32:898 (1968)). According to this model, amino acidresidues in a substrate undergoing cleavage are designated P1, P2, P3,P4 etc. in the N-terminal direction from the cleaved bond. Likewise, theresidues in the C-terminal direction are designated P1′, P2′, P3′, P4′.etc.

The skilled artisan is aware of a variety of tools for identifyingprotease binding or protease cleavage sites of interest. For example,the PeptideCutter software tool is available through the ExPASy (ExpertProtein Analysis System) proteomics server of the Swiss Institute ofBioinformatics (SIB; expasy.org/tools/peptidecutter). PeptideCuttersearches a protein sequence from the SWISS-PROT and/or TrEMBL databasesor a user-entered protein sequence for protease cleavage sites. Singleproteases and chemicals, a selection or the whole list of proteases andchemicals can be used. Different forms of output of the results areavailable: tables of cleavage sites either grouped alphabeticallyaccording to enzyme names or sequentially according to the amino acidnumber. A third option for output is a map of cleavage sites. Thesequence and the cleavage sites mapped onto it are grouped in blocks,the size of which can be chosen by the user. Other tools are also knownfor determining protease cleavage sites. (E.g., Turk, B. et al.,Determination of protease cleavage site motifs using mixture-basedoriented peptide libraries, Nature Biotechnology, 19:661-667 (2001);Barrett A. et al., Handbook of proteolytic enzymes, Academic Press(1998)).

The serine proteinases include the chymotrypsin family, which includesmammalian protease enzymes such as chymotrypsin, trypsin or elastase orkallikrein. The serine proteinases exhibit different substratespecificities, which are related to amino acid substitutions in thevarious enzyme subsites interacting with the substrate residues. Someenzymes have an extended interaction site with the substrate whereasothers have a specificity restricted to the P1 substrate residue.

Trypsin preferentially cleaves at R or K in position P1. A statisticalstudy carried out by Keil (1992) described the negative influences ofresidues surrounding the Arg- and Lys-bonds (i.e. the positions P2 andP1′, respectively) during trypsin cleavage. (Keil, B., Specificity ofproteolysis, Springer-Verlag Berlin-Heidelberg-New York, 335 (1992)). Aproline residue in position P1′ normally exerts a strong negativeinfluence on trypsin cleavage. Similarly, the positioning of R and K inP1′ results in an inhibition, as well as negatively charged residues inpositions P2 and P1′.

Chymotrypsin preferentially cleaves at a W, Y or F in position P1 (highspecificity) and to a lesser extent at L, M or H residue in position P1.(Keil, 1992). Exceptions to these rules are the following: When W isfound in position P1, the cleavage is blocked when M or P are found inposition P1′ at the same time. Furthermore, a proline residue inposition P1′ nearly fully blocks the cleavage independent of the aminoacids found in position P1. When an M residue is found in position P1,the cleavage is blocked by the presence of a Y residue in position P1′.Finally, when H is located in position P1, the presence of a D, M or Wresidue also blocks the cleavage.

Membrane metallo-endopeptidase (NEP; neutral endopeptidase,kidney-brush-border neutral proteinase, enkephalinase, EC 3.4.24.11)cleaves peptides at the amino side of hydrophobic amino acid residues.(Connelly, J C et al., Neutral Endopeptidase 24.11 in Human Neutrophils:Cleavage of Chemotactic Peptide, PNAS, 82(24):8737-8741 (1985)).

Thrombin preferentially cleaves at an R residue in position P1. (Keil,1992). The natural substrate of thrombin is fibrinogen. Optimum cleavagesites are when an R residue is in position P1 and Gly is in position P2and position P1′. Likewise, when hydrophobic amino acid residues arefound in position P4 and position P3, a proline residue in position P2,an R residue in position P1, and non-acidic amino acid residues inposition P1′ and position P2′. A very important residue for its naturalsubstrate fibrinogen is a D residue in P10.

Caspases are a family of cysteine proteases bearing an active site witha conserved amino acid sequence and which cleave peptides specificallyfollowing D residues. (Eamshaw W C et al., Mammalian caspases:Structure, activation, substrates, and functions during apoptosis,Annual Review of Biochemistry, 68:383-424 (1999)).

The Arg-C proteinase preferentially cleaves at an R residue in positionP1. The cleavage behavior seems to be only moderately affected byresidues in position P1′. (Keil, 1992). The Asp-N endopeptidase cleavesspecifically bonds with a D residue in position P1′. (Keil, 1992).

Furin is a ubiquitous subtilisin-like proprotein convertase. It is themajor processing enzyme of the secretory pathway and intracellularly islocalized in the trans-golgi network (van den Ouweland, A. M. W. et al.(1990) Nucl. Acids Res., 18, 664; Steiner, D. F. (1998) Curr. Opin.Chem. Biol., 2, 31-39). The minimal furin cleavage site is Arg-X-X-Arg′.However, the enzyme prefers the site Arg-X-(Lys/Arg)-Arg′. An additionalarginine at the P6 position appears to enhance cleavage (Krysan, D. J.et al. (1999) J. Biol. Chem., 274, 23229-23234).

The foregoing is merely exemplary and by no means an exhaustivetreatment of knowledge available to the skilled artisan concerningprotease binding and/or cleavage sites that the skilled artisan may beinterested in eliminating in practicing the invention.

Additional useful embodiments of the toxin peptide, e.g., the OSK1peptide analog, can result from conservative modifications of the aminoacid sequences of the peptides disclosed herein. Conservativemodifications will produce peptides having functional, physical, andchemical characteristics similar to those of the parent peptide fromwhich such modifications are made. Such conservatively modified forms ofthe peptides disclosed herein are also contemplated as being anembodiment of the present invention.

In contrast, substantial modifications in the functional and/or chemicalcharacteristics of the toxin peptides may be accomplished by selectingsubstitutions in the amino acid sequence that differ significantly intheir effect on maintaining (a) the structure of the molecular backbonein the region of the substitution, for example, as an α-helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the size of the molecule.

For example, a “conservative amino acid substitution” may involve asubstitution of a native amino acid residue with a normative residuesuch that there is little or no effect on the polarity or charge of theamino acid residue at that position. Furthermore, any native residue inthe polypeptide may also be substituted with alanine, as has beenpreviously described for “alanine scanning mutagenesis” (see, forexample, MacLennan et al., Acta Physiol. Scand. Suppl., 643:55-67(1998); Sasaki et al., 1998, Adv. Biophys. 35:1-24 (1998), which discussalanine scanning mutagenesis).

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. For example, amino acidsubstitutions can be used to identify important residues of the peptidesequence, or to increase or decrease the affinity of the peptide orvehicle-conjugated peptide molecules described herein.

Naturally occurring residues may be divided into classes based on commonside chain properties:

1) hydrophobic: norleucine (Nor), Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

3) acidic: Asp, Glu;

4) basic: His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

Conservative amino acid substitutions may involve exchange of a memberof one of these classes with another member of the same class.Conservative amino acid substitutions may encompass non-naturallyoccurring amino acid residues, which are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics and other reversed or invertedforms of amino acid moieties.

Non-conservative substitutions may involve the exchange of a member ofone of these classes for a member from another class. Such substitutedresidues may be introduced into regions of the human antibody that arehomologous with non-human antibodies, or into the non-homologous regionsof the molecule.

In making such changes, according to certain embodiments, thehydropathic index of amino acids may be considered. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics. They are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art(see, for example, Kyte et al., 1982, J. MOL Biol. 157:105-131). It isknown that certain amino acids may be substituted for other amino acidshaving a similar hydropathic index or score and still retain a similarbiological activity. In making changes based upon the hydropathic index,in certain embodiments, the substitution of amino acids whosehydropathic indices are within ±2 is included. In certain embodiments,those that are within ±1 are included, and in certain embodiments, thosewithin ±0.5 are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional protein or peptidethereby created is intended for use in immunological embodiments, asdisclosed herein. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5)and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, in certain embodiments, the substitution of aminoacids whose hydrophilicity values are within ±2 is included, in certainembodiments, those that are within ±1 are included, and in certainembodiments, those within ±0.5 are included. One may also identifyepitopes from primary amino acid sequences on the basis ofhydrophilicity. These regions are also referred to as “epitopic coreregions.”

Examples of conservative substitutions include the substitution of onenon-polar (hydrophobic) amino acid residue such as isoleucine, valine,leucine norleucine, alanine, or methionine for another, the substitutionof one polar (hydrophilic) amino acid residue for another such asbetween arginine and lysine, between glutamine and asparagine, betweenglycine and serine, the substitution of one basic amino acid residuesuch as lysine, arginine or histidine for another, or the substitutionof one acidic residue, such as aspartic acid or glutamic acid foranother. The phrase “conservative amino acid substitution” also includesthe use of a chemically derivatized residue in place of anon-derivatized residue, provided that such polypeptide displays therequisite biological activity. Other exemplary amino acid substitutionsthat can be useful in accordance with the present invention are setforth in Table 1A.

TABLE 1A Some Useful Amino Acid Substitutions Original ResiduesExemplary Substitutions Ala Val, Leu, Ile Arg Lys, Gln, Asn Asn Gln AspGlu Cys Ser, Ala Gln Asn Glu Asp Gly Pro, Ala His Asn, Gln, Lys, Arg IleLeu, Val, Met, Ala, Phe, Norleucine Leu Norleucine, Ile, Val, Met, Ala,Phe Lys Arg, 1,4-Diaminobutyric Acid, Gln, Asn Met Leu, Phe, Ile PheLeu, Val, Ile, Ala, Tyr Pro Ala Ser Thr, Ala, Cys Thr Ser Trp Tyr, PheTyr Trp, Phe, Thr, Ser Val Ile, Met, Leu, Phe, Ala, Norleucine

In other examples, a toxin peptide amino acid sequence, e.g., an OSK1peptide analog sequence, modified from a naturally occurring toxinpeptide amino acid sequence includes at least one amino acid residueinserted or substituted therein, relative to the amino acid sequence ofthe native toxin peptide sequence of interest, in which the inserted orsubstituted amino acid residue has a side chain comprising anucleophilic or electrophilic reactive functional group by which thepeptide is conjugated to a linker or half-life extending moiety. Inaccordance with the invention, useful examples of such a nucleophilic orelectrophilic reactive functional group include, but are not limited to,a thiol, a primary amine, a seleno, a hydrazide, an aldehyde, acarboxylic acid, a ketone, an aminooxy, a masked (protected) aldehyde,or a masked (protected) keto functional group. Examples of amino acidresidues having a side chain comprising a nucleophilic reactivefunctional group include, but are not limited to, a lysine residue, anα,β-diaminopropionic acid residue, an α,γ-diaminobutyric acid residue,an ornithine residue, a cysteine, a homocysteine, a glutamic acidresidue, an aspartic acid residue, or a selenocysteine residue. In someembodiments, the toxin peptide amino acid sequence (or “primarysequence”) is modified at one, two, three, four, five or more amino acidresidue positions, by having a residue substituted therein differentfrom the native primary sequence (e.g., OSK1 SEQ ID NO:25) or omitted(e.g., an OSK1 peptide analog optionally lacking a residue at positions36, 37, 36-38, 37-38, or 38).

In further describing toxin peptides herein, a one-letter abbreviationsystem is frequently applied to designate the identities of the twenty“canonical” amino acid residues generally incorporated into naturallyoccurring peptides and proteins (Table 1B). Such one-letterabbreviations are entirely interchangeable in meaning with three-letterabbreviations, or non-abbreviated amino acid names. Within theone-letter abbreviation system used herein, an uppercase letterindicates a L-amino acid, and a lower case letter indicates a D-aminoacid, unless otherwise noted herein. For example, the abbreviation “R”designates L-arginine and the abbreviation “r” designates D-arginine.

TABLE 1B One-letter abbreviations for the canonical amino acidsThree-letter abbreviations are in parentheses Alanine (Ala) A Glutamine(Gln) Q Leucine (Leu) L Serine (Ser) S Arginine (Arg) R Glutamic Acid(Glu) E Lysine (Lys) K Threonine (Thr) T Asparagine (Asn) N Glycine(Gly) G Methionine (Met) M Tryptophan (Trp) W Aspartic Acid (Asp) DHistidine (His) H Phenylalanine (Phe) F Tyrosine (Tyr) Y Cysteine (Cys)C Isoleucine (Ile) I Proline (Pro) P Valine (Val) V

An amino acid substitution in an amino acid sequence is typicallydesignated herein with a one-letter abbreviation for the amino acidresidue in a particular position, followed by the numerical amino acidposition relative to the native toxin peptide sequence of interest,which is then followed by the one-letter symbol for the amino acidresidue substituted in. For example, “T30D” symbolizes a substitution ofa threonine residue by an aspartate residue at amino acid position 30,relative to a hypothetical native toxin peptide sequence. By way offurther example, “R18hR” or “R18Cit” indicates a substitution of anarginine residue by a homoarginine or a citrulline residue,respectively, at amino acid position 18, relative to the hypotheticalnative toxin peptide. An amino acid position within the amino acidsequence of any particular toxin peptide (or peptide analog) describedherein may differ from its position relative to the native sequence,i.e., as determined in an alignment of the N-terminal or C-terminal endof the peptide's amino acid sequence with the N-terminal or C-terminalend, as appropriate, of the native toxin peptide sequence. For example,amino acid position 1 of the sequence SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTCShK(2-35); SEQ ID NO:92), a N-terminal truncation of the native ShKsequence, thus aligned with the C-terminal of native ShK(1-35) (SEQ IDNO:5), corresponds to amino acid position 2 relative to the nativesequence, and amino acid position 34 of SEQ ID NO:92 corresponds toamino acid position 35 relative to the native sequence (SEQ ID NO:5).

In certain embodiments of the present invention, amino acidsubstitutions encompass, non-canonical amino acid residues, whichinclude naturally rare (in peptides or proteins) amino acid residues orunnatural amino acid residues. Non-canonical amino acid residues can beincorporated into the peptide by chemical peptide synthesis rather thanby synthesis in biological systems, such as recombinantly expressingcells, or alternatively the skilled artisan can employ known techniquesof protein engineering that use recombinantly expressing cells. (See,e.g., Link et al., Non-canonical amino acids in protein engineering,Current Opinion in Biotechnology, 14(6):603-609 (2003)). The term“non-canonical amino acid residue” refers to amino acid residues in D-or L-form that are not among the 20 canonical amino acids generallyincorporated into naturally occurring proteins, for example, β-aminoacids, homoamino acids, cyclic amino acids and amino acids withderivatized side chains. Examples include (in the L-form or D-form;abbreviated as in parentheses): citrulline (Cit), homocitrulline (hCit),N^(α)-methylcitrulline (NMeCit), N^(α)-methylhomocitrulline(N^(α)-MeHoCit), ornithine (Orn), N^(α)-Methylornithine (N^(α)-MeOrn orNMeOrn), sarcosine (Sar), homolysine (hLys or hK), homoarginine (hArg orhR), homoglutamine (hQ), N^(α)-methylarginine (NMeR),N^(α)-methylleucine (N^(α)-MeL or NMeL), N-methylhomolysine (NMeHoK),N^(α)-methylglutamine (NMeQ), norleucine (Nle), norvaline (Nva),1,2,3,4-tetrahydroisoquinoline (Tic), Octahydroindole-2-carboxylic acid(Oic), 3-(1-naphthyl)alanine (1-Nal), 3-(2-naphthyl)alanine (2-Nal),1,2,3,4-tetrahydroisoquinoline (Tic), 2-indanylglycine (Igl),para-iodophenylalanine (pI-Phe), para-aminophenylalanine (4AmP or4-Amino-Phe), 4-guanidino phenylalanine (Guf), glycyllysine (abbreviatedherein “K(N^(∈)-glycyl)” or “K(glycyl)” or “K(gly)”), nitrophenylalanine(nitrophe), aminophenylalanine (aminophe or Amino-Phe),benzylphenylalanine (benzylphe), γ-carboxyglutamic acid (γ-carboxyglu),hydroxyproline (hydroxypro), p-carboxyl-phenylalanine (Cpa),α-aminoadipic acid (Aad), Nα-methyl valine (NMeVal), N-α-methyl leucine(NMeLeu), Nα-methylnorleucine (NMeNle), cyclopentylglycine (Cpg),cyclohexylglycine (Chg), acetylarginine (acetylarg),α,β-diaminopropionoic acid (Dpr), α,γ-diaminobutyric acid (Dab),diaminopropionic acid (Dap), cyclohexylalanine (Cha),4-methyl-phenylalanine (MePhe), β,β-diphenyl-alanine (BiPhA),aminobutyric acid (Abu), 4-phenyl-phenylalanine (or biphenylalanine;4Bip), α-amino-isobutyric acid (Aib), beta-alanine, beta-aminopropionicacid, piperidinic acid, aminocaproic acid, aminoheptanoic acid,aminopimelic acid, desmosine, diaminopimelic acid, N-ethylglycine,N-ethylaspargine, hydroxylysine, allo-hydroxylysine, isodesmosine,allo-isoleucine, N-methylglycine, N-methylisoleucine, N-methylvaline,4-hydroxyproline (Hyp), γ-carboxyglutamate, ∈-N,N,N-trimethyllysine,∈-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, ω-methylarginine, 4-Amino-O-PhthalicAcid (4APA), and other similar amino acids, and derivatized forms of anyof these as described herein. Table 1B contains some exemplarynon-canonical amino acid residues that are useful in accordance with thepresent invention and associated abbreviations as typically used herein,although the skilled practitioner will understand that differentabbreviations and nomenclatures may be applicable to the same substanceand my appear interchangeably herein.

Table 1B. Useful non-canonical amino acids for amino acid addition,insertion, or substitution into toxin peptide sequences, including OSK1peptide analog sequences, in accordance with the present invention. Inthe event an abbreviation listed in Table 1B differs from anotherabbreviation for the same substance disclosed elsewhere herein, bothabbreviations are understood to be applicable.

Abbreviation Amino Acid Sar Sarcosine Nle norleucine Ile isoleucine1-Nal 3-(1-naphthyl)alanine 2-Nal 3-(2-naphthyl)alanine Bip4,4′-biphenyl alanine Dip 3,3-diphenylalanine Nvl norvaline NMe-ValNα-methyl valine NMe-Leu Nα-methyl leucine NMe-Nle Nα-methyl norleucineCpg cyclopentyl glycine Chg cyclohexyl glycine Hyp hydroxy proline OicOctahydroindole-2-Carboxylic Acid Igl Indanyl glycine Aibaminoisobutyric acid Aic 2-aminoindane-2-carboxylic acid Pip pipecolicacid BhTic β-homo Tic BhPro β-homo proline Sar Sarcosine Cpg cyclopentylglycine Tiq 1,2,3,4-L-Tetrahydroisoquinoline-1-Carboxylic acid NipNipecotic Acid Thz Thiazolidine-4-carboxylic acid Thi 3-thienyl alanine4GuaPr 4-guanidino proline 4Pip 4-Amino-1-piperidine-4-carboxylic acidIdc indoline-2-carboxylic acid Hydroxyl-Tic1,2,3,4-Tetrahydroisoquinoline-7-hydroxy-3- carboxylic acid Bip4,4′-biphenyl alanine Ome-Tyr O-methyl tyrosine I-Tyr Iodotyrosine Tic1,2,3,4-L-Tetrahydroisoquinoline-3-Carboxylic acid Igl Indanyl glycineBhTic β-homo Tic BhPhe β-homo phenylalanine AMeF α-methyl PhenyalanineBPhe β-phenylalanine Phg phenylglycine Anc 3-amino-2-naphthoic acid Atc2-aminotetraline-2-carboxylic acid NMe-Phe Nα-methyl phenylalanineNMe-Lys Nα-methyl lysine Tpi 1,2,3,4-Tetrahydronorharman-3-Carboxylicacid Cpg cyclopentyl glycine Dip 3,3-diphenylalanine 4Pal4-pyridinylalanine 3Pal 3-pyridinylalanine 2Pal 2-pyridinylalanine 4Pip4-Amino-1-piperidine-4-carboxylic acid 4AmP 4-amino-phenylalanine Idcindoline-2-carboxylic acid Chg cyclohexyl glycine hPhe homophenylalanineBhTrp β-homotryptophan pI-Phe 4-iodophenylalanine Aic2-aminoindane-2-carboxylic acid NMe-Lys Nα-methyl lysine Orn ornithineDpr 2,3-Diaminopropionic acid Dbu 2,4-Diaminobutyric acid homoLyshomolysine N-eMe-K Nε-methyl-lysine N-eEt-K Nε-ethyl-lysine N-eIPr-KNε-isopropyl-lysine bhomoK β-homolysine rLys Lys ψ (CH₂NH)-reduced amidebond rOrn Orn ψ (CH₂NH)-reduced amide bond Acm acetamidomethyl Ahx6-aminohexanoic acid ε Ahx 6-aminohexanoic acid K(NPeg11)Nε-(O-(aminoethyl)-O′-(2-propanoyl)- undecaethyleneglycol)-LysineK(NPeg27) Nε-(O-(aminoethyl)-O′-(2-propanoyl)- (ethyleneglycol)27-LysineCit Citrulline hArg homoarginine hCit homocitrulline NMe-Arg Nα-methylarginine (NMeR) Guf 4-guanidinyl phenylalanine bhArg β-homoarginine3G-Dpr 2-amino-3-guanidinopropanoic acid 4AmP 4-amino-phenylalanine4AmPhe 4-amidino-phenylalanine 4AmPig2-amino-2-(1-carbamimidoylpiperidin-4- yl)acetic acid 4GuaPr 4-guanidinoproline N-Arg Nα-[(CH₂)₃NHCH(NH)NH₂] substituted glycine rArg Argψ(CH₂NH) -reduced amide bond 4PipA 4-Piperidinyl alanine NMe-ArgNα-methyl arginine (or NMeR) NMe-Thr Nα-methyl threonine(or NMeThr)

Nomenclature and Symbolism for Amino Acids and Peptides by the UPAC-IUBJoint Commission on Biochemical Nomenclature (JCBN) have been publishedin the following documents: Biochem. J., 1984, 219, 345-373; Eur. J.Biochem., 1984, 138, 9-37; 1985, 152, 1; 1993, 213, 2; Internat. J.Pept. Prot. Res., 1984, 24, following p 84; J. Biol. Chem., 1985, 260,14-42; Pure Appl. Chem., 1984, 56, 595-624; Amino Acids and Peptides,1985, 16, 387-410; Biochemical Nomenclature and Related Documents, 2ndedition, Portland Press, 1992, pages 39-69].

As stated herein, in accordance with the present invention, peptideportions of the inventive compositions, such as the toxin peptide or apeptide linker, can also be chemically derivatized at one or more aminoacid residues. Peptides that contain derivatized amino acid residues canbe synthesized by known organic chemistry techniques. “Chemicalderivative” or “chemically derivatized” in the context of a peptiderefers to a subject peptide having one or more residues chemicallyderivatized by reaction of a functional side group. Such derivatizedmolecules include, for example, those molecules in which free aminogroups have been derivatized to form amine hydrochlorides, p-toluenesulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups,chloroacetyl groups or formyl groups. Free carboxyl groups may bederivatized to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups may be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine maybe derivatized to form N-im-benzylhistidine. Also included as chemicalderivatives are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty canonical amino acids,whether in L- or D-form. For example, 4-hydroxyproline may besubstituted for proline; 5-hydroxylysine maybe substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.

Useful derivatizations include, in some embodiments, those in which theamino terminal of the toxin peptide, such as but not limited to the OSK1peptide analog, is chemically blocked so that conjugation with thevehicle will be prevented from taking place at an N-terminal free aminogroup. There may also be other beneficial effects of such amodification, for example a reduction in the toxin peptide'ssusceptibility to enzymatic proteolysis. The N-terminus of the toxinpeptide, e.g., the OSK1 peptide analog, can be acylated or modified to asubstituted amine, or derivatized with another functional group, such asan aromatic or aryl moiety (e.g., an indole acid, benzyl (Bzl or Bn),dibenzyl (DiBzl or Bn₂), benzoyl, or benzyloxycarbonyl (Cbz or Z)),N,N-dimethylglycine or creatine. For example, in some embodiments, anacyl moiety, such as, but not limited to, a formyl, acetyl (Ac),propanoyl, butanyl, heptanyl, hexanoyl, octanoyl, or nonanoyl, can becovalently linked to the N-terminal end of the peptide, e.g., the OSK1peptide analog, which can prevent undesired side reactions duringconjugation of the vehicle to the peptide. Alternatively, a fatty acid(e.g. butyric, caproic, caprylic, capric, lauric, myristic, palmitic,stearic or the like) or polyethylene glycol moiety can be covalentlylinked to the N-terminal end of the peptide, e.g., the OSK1 peptideanalog. Other exemplary N-terminal derivative groups include —NRR¹(other than —NH₂), —NRC(O)R¹, —NRC(O)OR¹, —NRS(O)₂R¹, —NHC(O)NHR¹,succinimide, or benzyloxycarbonyl-NH-(Cbz-NH—), wherein R and R¹ areeach independently hydrogen or lower alkyl and wherein the phenyl ringmay be substituted with 1 to 3 substituents selected from C₁-C₄ alkyl,C₁-C₄ alkoxy, chloro, and bromo.

In some embodiments of the present invention, basic residues (e.g.,lysine) of the toxin peptide of interest can be replaced with otherresidues (nonfunctional residues preferred). Such molecules will be lessbasic than the molecules from which they are derived and otherwiseretain the activity of the molecules from which they are derived, whichcan result in advantages in stability and immunogenicity; the presentinvention should not, however, be limited by this theory.

Additionally, physiologically acceptable salts of the inventivecompositions are also encompassed, including when the inventivecompositions are referred to herein as “molecules” or “compounds.”. By“physiologically acceptable salts” is meant any salts that are known orlater discovered to be pharmaceutically acceptable. Some non-limitingexamples of pharmaceutically acceptable salts are: acetate;trifluoroacetate; hydrohalides, such as hydrochloride and hydrobromide;sulfate; citrate; maleate; tartrate; glycolate; gluconate; succinate;mesylate; besylate; salts of gallic acid esters (gallic acid is alsoknown as 3, 4, 5 trihydroxybenzoic acid) such as PentaGalloylGlucose(PGG) and epigallocatechin gallate (EGCG), salts of cholesteryl sulfate,pamoate, tannate and oxalate salts.

Structure of Compounds:

In general. Recombinant proteins have been developed as therapeuticagents through, among other means, covalent attachment to half-lifeextending moieties. Such moieties include the “Fc” domain of anantibody, as is used in Enbrel® (etanercept), as well as biologicallysuitable polymers (e.g., polyethylene glycol, or “PEG”), as is used inNeulasta® (pegfilgrastim). Feige et al. described the use of suchhalf-life extenders with peptides in U.S. Pat. No. 6,660,843, issuedDec. 9, 2003 (hereby incorporated by reference in its entirety).

The present inventors have determined that molecules of thisinvention-peptides of about 80 amino acids or less with at least twointrapeptide disulfide bonds-possess therapeutic advantages whencovalently attached to half-life extending moieties. Molecules of thepresent invention can further comprise an additional pharmacologicallyactive, covalently bound peptide, which can be bound to the half-lifeextending moiety (F¹ and/or F²) or to the peptide portion (P).Embodiments of the inventive compositions containing more than onehalf-life extending moiety (F¹ and F²) include those in which F¹ and F²are the same or different half-life extending moieties. Examples (withor without a linker between each domain) include structures asillustrated in FIG. 75 as well as the following embodiments (and othersdescribed herein and in the working Examples):

20KPEG—toxin peptide—Fc domain, consistent with the formula[(F¹)₁-(X²)₁-(F²)₁];

20KPEG—toxin peptide—Fc CH2 domain, consistent with the formula[(F¹)₁-(X²)₁-(F²)₁];

20KPEG—toxin peptide—HSA, consistent with the formula[(F¹)₁(X²)₁-(F²)₁];

20KPEG—Fc domain—toxin peptide, consistent with the formula[(F¹)₁-(F²)₁-(X³)₁];

20KPEG—Fc CH2 domain—toxin peptide, consistent with the formula[(F¹)₁-(F²)₁-(X³)₁]; and

20KPEG—HSA—toxin peptide, consistent with the formula[(F¹)₁-(F²)₁-(X³)₁].

Toxin peptides. Any number of toxin peptides (i.e., “P”, or equivalentlyshown as “P¹” in FIG. 2) can be used in conjunction with the presentinvention. Of particular interest are the toxin peptides ShK, HmK, MgTx,AgTx2, Agatoxins, and HsTx1, as well as modified analogs of these, inparticular OsK1 (also referred to as “OSK1”) peptide analogs of thepresent invention, and other peptides that mimic the activity of suchtoxin peptides. As stated herein above, if more than one toxin peptide“P” is present in the inventive composition, “P” can be independentlythe same or different from any other toxin peptide(s) also present inthe inventive composition. For example, in a composition having theformula P-(L)_(g)-F¹-(L)_(f)-P, both of the toxin peptides, “P”, can bethe same peptide analog of ShK, different peptide analogs of ShK, or onecan be a peptide analog of ShK and the other a peptide analog of OSK1.In a preferred embodiment, at least one P is a an OSK1 peptide analog asfurther described herein.

In some embodiments of the invention, other peptides of interest areespecially useful in molecules having additional features over themolecules of structural Formula I. In such molecules, the molecule ofFormula I further comprises an additional pharmacologically active,covalently bound peptide, which is an agonistic peptide, an antagonisticpeptide, or a targeting peptide; this peptide can be conjugated to F¹ orF² or P. Such agonistic peptides have activity agonistic to the toxinpeptide but are not required to exert such activity by the samemechanism as the toxin peptide. Peptide antagonists are also useful inembodiments of the invention, with a preference for those with activitythat can be complementary to the activity of the toxin peptide.Targeting peptides are also of interest, such as peptides that directthe molecule to particular cell types, organs, and the like. Theseclasses of peptides can be discovered by methods described in thereferences cited in this specification and other references. Phagedisplay, in particular, is useful in generating toxin peptides for usein the present invention. Affinity selection from libraries of randompeptides can be used to identify peptide ligands for any site of anygene product. Dedman et al. (1993), J. Biol. Chem. 268: 23025-30. Phagedisplay is particularly well suited for identifying peptides that bindto such proteins of interest as cell surface receptors or any proteinshaving linear epitopes. Wilson et al. (1998), Can. J. Microbiol. 44:313-29; Kay et al. (1998), Drug Disc. Today 3: 370-8. Such proteins areextensively reviewed in Herz et al. (1997), J. Receptor and SignalTransduction Res. 17(5): 671-776, which is hereby incorporated byreference in its entirety. Such proteins of interest are preferred foruse in this invention.

Particularly preferred peptides appear in the following tables. Thesepeptides can be prepared by methods disclosed in the art or as describedhereinafter. Single letter amino acid abbreviations are used. Unlessotherwise specified, each X is independently a nonfunctional residue.

TABLE 1 Kv1.3 inhibitor peptide sequences SEQ Short-hand IDSequence/structure designation NO: LVKCRGTSDCGRPCQQQTGCPNSKCINRMCKCYGCPi1 21 TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFGR Pi2 17TISCTNEKQCYPHCKKETGYPNAKCMNRKCKCFGR Pi3 18IEAIRCGGSRDCYRPCQKRTGCPNAKCINKTCKCY Pi4 19 GCSASCRTPKDCADPCRKETGCPYGKCMNRKCKCNRC HsTx1 61GVPINVSCTGSPQCIKPCKDAGMRFGKCMNRKCHC AgTx2 23 TPKGVPINVKCTGSPQCLKPCKDAGMRFGKCINGKCHC AgTx1 85 TPKGVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC OSK1 25 TPKZKECTGPQHCTNFCRKNKCTHGKCMNRKCKCFNCK Anuroctoxin 62TIINVKCTSPKQCSKPCKELYGSSAGAKCMNGKCK NTX 30 CYNNTVIDVKCTSPKQCLPPCKAQFGIRAGAKCMNGKCK HgTx1 27 CYPHQFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRC ChTx 36 YSVFINAKCRGSPECLPKCKEAIGKAAGKCMNGKCKC Titystoxin- 86 YP KaVCRDWFKETACRHAKSLGNCRTSQKYRANCAKTCE BgK 9 LCVGINVKCKHSGQCLKPCKDAGMRFGKCINGKCDCT BmKTX 26 PKGQFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCRC BmTx1 40 YSVFINVKCRGSKECLPACKAAVGKAAGKCMNGKCKC Tc30 87 YPTGPQTTCQAAMCEAGCKGLGKSMESCQGDTCKCKA Tc32 13

TABLE 2 ShK peptide and ShK peptide analog sequences Short-hand SEQSequence/structure designation ID NO:RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK 5RSCIDTIPKSRCTAFQSKHSMKYRLSFCRKTSGTC ShK-S17/S32 88RSSIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTS ShK-S3/S35 89SSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-S1 90 (N-acetylarg)ShK-N-acetylarg1 91 SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-d1 92  CIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-d2 93ASCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A1 94RSCADTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A4 95RSCADTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC ShK-A4/A15 96RSCADTIPKSRCTAAQCKHSMKYRASFCRKTCGTC ShK-A4/A15/A25 97RSCIDAIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A6 98RSCIDTAPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A7 99RSCIDTIAKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A8 100RSCIDTIPASRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A9 101RSCIDTIPESRCTAFQCKHSMKYRLSFCRKTCGTC ShK-E9 102RSCIDTIPQSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-Q9 103RSCIDTIPKARCTAFQCKHSMKYRLSFCRKTCGTC ShK-A10 104RSCIDTIPKSACTAFQCKHSMKYRLSFCRKTCGTC ShK-A11 105RSCIDTIPKSECTAFQCKHSMKYRLSFCRKTCGTC ShK-E11 106RSCIDTIPKSQCTAFQCKHSMKYRLSFCRKTCGTC ShK-Q11 107RSCIDTIPKSRCAAFQCKHSMKYRLSFCRKTCGTC ShK-A13 108RSCIDTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC ShK-A15 109RSCIDTIPKSRCTAWQCKHSMKYRLSFCRKTCGTC ShK-W15 110RSCIDTIPKSRCTAX^(s15)QCKHSMKYRLSFCRKTCGTC ShK-X15 111RSCIDTIPKSRCTAAQCKHSMKYRASFCRKTCGTC ShK-A15/A25 112RSCIDTIPKSRCTAFACKHSMKYRLSFCRKTCGTC ShK-A16 113RSCIDTIPKSRCTAFECKHSMKYRLSFCRKTCGTC ShK-E16 114RSCIDTIPKSRCTAFQCAHSMKYRLSFCRKTCGTC ShK-A18 115RSCIDTIPKSRCTAFQCEHSMKYRLSFCRKTCGTC ShK-E18 116RSCIDTIPKSRCTAFQCKASMKYRLSFCRKTCGTC ShK-A19 117RSCIDTIPKSRCTAFQCKKSMKYRLSFCRKTCGTC ShK-K19 118RSCIDTIPKSRCTAFQCKHAMKYRLSFCRKTCGTC ShK-A20 119RSCIDTIPKSRCTAFQCKHSAKYRLSFCRKTCGTC ShK-A21 120RSCIDTIPKSRCTAFQCKHSX^(s21)KYRLSFCRKTCGTC ShK-X21 121RSCIDTIPKSRCTAFQCKHS(norleu)KYRLSFCRKTCGTC ShK-Nle21 122RSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC ShK-A22 123RSCIDTIPKSRCTAFQCKHSMEYRLSFCRKTCGTC ShK-E22 124RSCIDTIPKSRCTAFQCKHSMRYRLSFCRKTCGTC ShK-R22 125RSCIDTIPKSRCTAFQCKHSMX^(s22)YRLSFCRKTCGTC ShK-X22 126RSCIDTIPKSRCTAFQCKHSM(norleu)YRLSFCRKTCGTC ShK-Nle22 127RSCIDTIPKSRCTAFQCKHSM(orn)YRLSFCRKTCGTC ShK-Orn22 128RSCIDTIPKSRCTAFQCKHSM(homocit)YRLSFCRKTCGTC ShK-Homocit22 129RSCIDTIPKSRCTAFQCKHSM(diaminopropionic)YRLS ShK-Diamino- 130 FCRKTCGTCpropionic22 RSCIDTIPKSRCTAFQCKHSMKARLSFCRKTCGTC ShK-A23 131RSCIDTIPKSRCTAFQCKHSMKSRLSFCRKTCGTC ShK-S23 132RSCIDTIPKSRCTAFQCKHSMKFRLSFCRKTCGTC ShK-F23 133RSCIDTIPKSRCTAFQCKHSMKX^(s23)RLSFCRKTCGTC ShK-X23 134RSCIDTIPKSRCTAFQCKHSMK(nitrophe)RLSFCRKTCGTC ShK-Nitrophe23 135RSCIDTIPKSRCTAFQCKHSMK(aminophe)RLSFCRKTCGTC ShK-Aminophe23 136RSCIDTIPKSRCTAFQCKHSMK(benzylphe)RLSFCRKTCG ShK-Benzylphe23 137 TCRSCIDTIPKSRCTAFQCKHSMKYALSFCRKTCGTC ShK-A24 138RSCIDTIPKSRCTAFQCKHSMKYELSFCRKTCGTC ShK-E24 139RSCIDTIPKSRCTAFQCKHSMKYRASFCRKTCGTC ShK-A25 140RSCIDTIPKSRCTAFQCKHSMKYRLAFCRKTCGTC ShK-A26 141RSCIDTIPKSRCTAFQCKHSMKYRLSACRKTCGTC ShK-A27 142RSCIDTIPKSRCTAFQCKHSMKYRLSX^(s27)CRKTCGTC ShK-X27 143RSCIDTIPKSRCTAFQCKHSMKYRLSFCAKTCGTC ShK-A29 144RSCIDTIPKSRCTAFQCKHSMKYRLSFCRATCGTC ShK-A30 145RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKACGTC ShK-A31 146RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGAC ShK-A34 147 SCADTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A4d1 148 SCADTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC ShK-A4/A15d1 149 SCADTIPKSRCTAAQCKHSMKYRASFCRKTCGTC ShK-A4/A15/A25 150 d1 SCIDAIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A6 d1 151 SCIDTAPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A7 d1 152 SCIDTIAKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A8 d1 153 SCIDTIPASRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A9 d1 154 SCIDTIPESRCTAFQCKHSMKYRLSFCRKTCGTC ShK-E9 d1 155 SCIDTIPQSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-Q9 d1 156 SCIDTIPKARCTAFQCKHSMKYRLSFCRKTCGTC ShK-A10 d1 157 SCIDTIPKSACTAFQCKHSMKYRLSFCRKTCGTC ShK-A11 d1 158 SCIDTIPKSECTAFQCKHSMKYRLSFCRKTCGTC ShK-E11 d1 159 SCIDTIPKSQCTAFQCKHSMKYRLSFCRKTCGTC ShK-Q11 d1 160 SCIDTIPKSRCAAFQCKHSMKYRLSFCRKTCGTC ShK-A13 d1 161 SCIDTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC ShK-A15 d1 162 SCIDTIPKSRCTAWQCKHSMKYRLSFCRKTCGTC ShK-W15 d1 163 SCIDTIPKSRCTAX^(s15)QCKHSMKYRLSFCRKTCGTC ShK-X15 d1 164 SCIDTIPKSRCTAAQCKHSMKYRASFCRKTCGTC ShK-A15/A25 d1 165 SCIDTIPKSRCTAFACKHSMKYRLSFCRKTCGTC ShK-A16 d1 166 SCIDTIPKSRCTAFECKHSMKYRLSFCRKTCGTC ShK-E16 d1 167 SCIDTIPKSRCTAFQCAHSMKYRLSFCRKTCGTC ShK-A18 d1 168 SCIDTIPKSRCTAFQCEHSMKYRLSFCRKTCGTC ShK-E18 d1 169 SCIDTIPKSRCTAFQCKASMKYRLSFCRKTCGTC ShK-A19 d1 170 SCIDTIPKSRCTAFQCKKSMKYRLSFCRKTCGTC ShK-K19 d1 171 SCIDTIPKSRCTAFQCKHAMKYRLSFCRKTCGTC ShK-A20 d1 172 SCIDTIPKSRCTAFQCKHSAKYRLSFCRKTCGTC ShK-A21 d1 173 SCIDTIPKSRCTAFQCKHSX^(s21)KYRLSFCRKTCGTC ShK-X21 d1 174SCIDTIPKSRCTAFQCKHS(norleu)KYRLSFCRKTCGTC ShK-Nle21 d1 175 SCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC ShK-A22 d1 176 SCIDTIPKSRCTAFQCKHSMEYRLSFCRKTCGTC ShK-E22 d1 177 SCIDTIPKSRCTAFQCKHSMRYRLSFCRKTCGTC ShK-R22 d1 178 SCIDTIPKSRCTAFQCKHSMX^(s22)YRLSFCRKTCGTC ShK-X22 d1 179SCIDTIPKSRCTAFQCKHSM(norleu)YRLSFCRKTCGTC ShK-Nle22 d1 180SCIDTIPKSRCTAFQCKHSM(orn)YRLSFCRKTCGTC ShK-Orn22 d1 181SCIDTIPKSRCTAFQCKHSM(homocit)YRLSFCRKTCGTC ShK-Homocit22 182 d1SCIDTIPKSRCTAFQCKHSM(diaminopropionic)YRLSF ShK-Diamino- 183 CRKTCGTCpropionic22 d1  SCIDTIPKSRCTAFQCKHSMKARLSFCRKTCGTC ShK-A23 d1 184 SCIDTIPKSRCTAFQCKHSMKSRLSFCRKTCGTC ShK-S23 d1 185 SCIDTIPKSRCTAFQCKHSMKFRLSFCRKTCGTC ShK-F23 d1 186 SCIDTIPKSRCTAFQCKHSMKX^(s23)RLSFCRKTCGTC ShK-X23 d1 187SCIDTIPKSRCTAFQCKHSMK(nitrophe)RLSFCRKTCGTC ShK-Nitrophe23 188 d1SCIDTIPKSRCTAFQCKHSMK(aminophe)RLSFCRKTCGTC ShK-Aminophe23 189 d1SCIDTIPKSRCTAFQCKHSMK(benzylphe)RLSFCRKTCGTC ShK-Benzylphe23 190 d1 SCIDTIPKSRCTAFQCKHSMKYALSFCRKTCGTC ShK-A24 d1 191 SCIDTIPKSRCTAFQCKHSMKYELSFCRKTCGTC ShK-E24 d1 192 SCIDTIPKSRCTAFQCKHSMKYRASFCRKTCGTC ShK-A25 d1 193 SCIDTIPKSRCTAFQCKHSMKYRLAFCRKTCGTC ShK-A26 d1 194 SCIDTIPKSRCTAFQCKHSMKYRLSACRKTCGTC ShK-A27 d1 195 SCIDTIPKSRCTAFQCKHSMKYRLSX^(s27)CRKTCGTC ShK-X27 d1 196 SCIDTIPKSRCTAFQCKHSMKYRLSFCAKTCGTC ShK-A29 d1 197 SCIDTIPKSRCTAFQCKHSMKYRLSFCRATCGTC ShK-A30 d1 198 SCIDTIPKSRCTAFQCKHSMKYRLSFCRKACGTC ShK-A31 d1 199 SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGAC ShK-A34 d1 200YSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-Y1 548KSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-K1 549HSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-H1 550QSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-Q1 551PPRSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC PP-ShK 552MRSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC M-ShK 553GRSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC G-ShK 554YSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC ShK-Y1/A22 555KSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC ShK-K1/A22 556HSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC ShK-H1/A22 557QSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC ShK-Q1/A22 558PPRSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC PP-ShK-A22 559MRSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC M-ShK-A22 560GRSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC G-ShK-A22 561RSCIDTIPASRCTAFQCKHSMAYRLSFCRKTCGTC ShK-A9/A22 884SCIDTIPASRCTAFQCKHSMAYRLSFCRKTCGTC ShK-A9/A22 d1 885RSCIDTIPVSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-V9 886RSCIDTIPVSRCTAFQCKHSMAYRLSFCRKTCGTC ShK-V9/A22 887SCIDTIPVSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-V9 d1 888SCIDTIPVSRCTAFQCKHSMAYRLSFCRKTCGTC ShK-V9/A22 d1 889RSCIDTIPESRCTAFQCKHSMAYRLSFCRKTCGTC ShK-E9/A22 890SCIDTIPESRCTAFQCKHSMAYRLSFCRKTCGTC ShK-E9/A22 d1 891RSCIDTIPKSACTAFQCKHSMAYRLSFCRKTCGTC ShK-A11/A22 892SCIDTIPKSACTAFQCKHSMAYRLSFCRKTCGTC ShK-A11/22 d1 893RSCIDTIPKSECTAFQCKHSMAYRLSFCRKTCGTC ShK-E11/A22 894SCIDTIPKSECTAFQCKHSMAYRLSFCRKTCGTC ShK-E11/A22 d1 895RSCIDTIPKSRCTDFQCKHSMKYRLSFCRKTCGTC ShK-D14 896RSCIDTIPKSRCTDFQCKHSMAYRLSFCRKTCGTC ShK-D14/A22 897SCIDTIPKSRCTDFQCKHSMKYRLSFCRKTCGTC ShK-D14 d1 898SCIDTIPKSRCTDFQCKHSMAYRLSFCRKTCGTC ShK-D14/A22 d1 899RSCIDTIPKSRCTAAQCKHSMAYRLSFCRKTCGTC ShK-A15A/22 900SCIDTIPKSRCTAAQCKHSMAYRLSFCRKTCGTC ShK-A15/A22 d1 901RSCIDTIPKSRCTAIQCKHSMKYRLSFCRKTCGTC ShK-I15 902RSCIDTIPKSRCTAIQCKHSMAYRLSFCRKTCGTC ShK-I15/A22 903SCIDTIPKSRCTAIQCKHSMKYRLSFCRKTCGTC ShK-I15 d1 904SCIDTIPKSRCTAIQCKHSMAYRLSFCRKTCGTC ShK-I15/A22 d1 905RSCIDTIPKSRCTAVQCKHSMKYRLSFCRKTCGTC ShK-V15 906RSCIDTIPKSRCTAVQCKHSMAYRLSFCRKTCGTC ShK-V15/A22 907SCIDTIPKSRCTAVQCKHSMKYRLSFCRKTCGTC ShK-V15 d1 908SCIDTIPKSRCTAVQCKHSMAYRLSFCRKTCGTC ShK-V15/A22 d1 909RSCIDTIPKSRCTAFRCKHSMKYRLSFCRKTCGTC ShK-R16 910RSCIDTIPKSRCTAFRCKHSMAYRLSFCRKTCGTC ShK-R16/A22 911SCIDTIPKSRCTAFRCKHSMKYRLSFCRKTCGTC ShK-R16 d1 912SCIDTIPKSRCTAFRCKHSMAYRLSFCRKTCGTC ShK-R16/A22 d1 913RSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC ShK-K16 914RSCIDTIPKSRCTAFKCKHSMAYRLSFCRKTCGTC ShK-K16/A22 915SCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC ShK-K16 d1 916SCIDTIPKSRCTAFKCKHSMAYRLSFCRKTCGTC ShK-K16/A22 d1 917RSCIDTIPASECTAFQCKHSMKYRLSFCRKTCGTC ShK-A9/E11 918RSCIDTIPASECTAFQCKHSMAYRLSFCRKTCGTC ShK-A9/E11/A22 919SCIDTIPASECTAFQCKHSMKYRLSFCRKTCGTC ShK-A9/E11 d1 920SCIDTIPASECTAFQCKHSMAYRLSFCRKTCGTC ShK-A9/E11/A22 921 d1RSCIDTIPVSECTAFQCKHSMKYRLSFCRKTCGTC ShK-V9/E11 922RSCIDTIPVSECTAFQCKHSMAYRLSFCRKTCGTC ShK-V9/E11/A22 923SCIDTIPVSECTAFQCKHSMKYRLSFCRKTCGTC ShK-V9/E11 d1 924SCIDTIPVSECTAFQCKHSMAYRLSFCRKTCGTC ShK-V9/E11/A22 925 d1RSCIDTIPVSACTAFQCKHSMKYRLSFCRKTCGTC ShK-V9/A11 926RSCIDTIPVSACTAFQCKHSMAYRLSFCRKTCGTC ShK-V9/A11/A22 927SCIDTIPVSACTAFQCKHSMKYRLSFCRKTCGTC ShK-V9/A11 d1 928SCIDTIPVSACTAFQCKHSMAYRLSFCRKTCGTC ShK-V9/A11/A22 929 d1RSCIDTIPASACTAFQCKHSMKYRLSFCRKTCGTC ShK-A9/A11 930RSCIDTIPASACTAFQCKHSMAYRLSFCRKTCGTC ShK-A9/A11/A22 931SCIDTIPASACTAFQCKHSMKYRLSFCRKTCGTC ShK-A9/A11 d1 932SCIDTIPASACTAFQCKHSMAYRLSFCRKTCGTC ShK-A9/A11/A22 933 d1RSCIDTIPKSECTDIRCKHSMKYRLSFCRKTCGTC ShK- 934 E11/D14/I15/R16RSCIDTIPKSECTDIRCKHSMAYRLSFCRKTCGTC ShK- 935 E11/D14/I15/R16/ A22SCIDTIPKSECTDIRCKHSMKYRLSFCRKTCGTC ShK- 936 E11/D14/I15/R16 d1SCIDTIPKSECTDIRCKHSMAYRLSFCRKTCGTC ShK- 937 E11/D14/I15//R16 A22 d1RSCIDTIPVSECTDIRCKHSMKYRLSFCRKTCGTC ShK- 938 V9/E11/D14/I15/ R16RSCIDTIPVSECTDIRCKHSMAYRLSFCRKTCGTC ShK- 939 V9/E11/D14/I15/ R16/A22SCIDTIPVSECTDIRCKHSMKYRLSFCRKTCGTC ShK- 940 V9/E11/D14/I15/ R16 d1SCIDTIPVSECTDIRCKHSMAYRLSFCRKTCGTC ShK- 941 V9/E11/D14/I15/ R16/A22 d1RSCIDTIPVSECTDIQCKHSMKYRLSFCRKTCGTC ShK- 942 V9/E11/D14/I15RSCIDTIPVSECTDIQCKHSMAYRLSFCRKTCGTC ShK- 943 V9/E11/D14/I15/A22SCIDTIPVSECTDIQCKHSMKYRLSFCRKTCGTC ShK- 944 V9/E11/D14/I15 d1SCIDTIPVSECTDIQCKHSMAYRLSFCRKTCGTC ShK- 945 V9/E11/D14/I15/A 22 d1RTCKDLIPVSECTDIRCKHSMKYRLSFCRKTCGTC ShK- 946 T2/K4/L6/V9/E11/D14/I15/R16 RTCKDLIPVSECTDIRCKHSMAYRLSFCRKTCGTC ShK- 947T2/K4/L6/V9/E11/ D14/I15/R16/A22 TCKDLIPVSECTDIRCKHSMKYRLSFCRKTCGTC ShK-948 T2/K4/L6/V9/E11/ D14/I15/R16 d1 TCKDLIPVSECTDIRCKHSMAYRLSFCRKTCGTCShK- 949 T2/K4/L6/V9/E11/ D14/I15/R16/A22 d1 (L-Phosphotyrosine)-ShK(L5) 950 AEEARSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTCQSCADTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC ShK Q1/A4/A15 1295QSCADTIPKSRCTAAQCKHSMAYRLSFCRKTCGTC ShK 1296 Q1/A4/A15/A22QSCADTIPKSRCTAAQCKHSM(Dap)YRLSFCRKTCGTC ShK 1297 Q1/A4/A15/Dap22QSCADTIPKSRCTAAQCKHSMKYRASFCRKTCGTC ShK 1298 Q1/A4/A15/A25QSCADTIPKSRCTAAQCKHSMAYRASFCRKTCGTC ShK 1299 Q1/A4/A15/A22/A25QSCADTIPKSRCTAAQCKHSM(Dap)YRASFCRKTCGTC ShK 1300 Q1/A4/A15/Dap22/ A25

Many peptides as described in Table 2 can be prepared as described inU.S. Pat. No. 6,077,680 issued Jun. 20, 2000 to Kem et al., which ishereby incorporated by reference in its entirety. Other peptides ofTable 2 can be prepared by techniques known in the art. For example,ShK(L5) (SEQ ID NO: 950) can be prepared as described in Beeton et al.,Targeting effector memory T cells with a selective peptide inhibitor ofKv1.3 channels for therapy of autoimmune diseases, Molec. Pharmacol.67(4): 1369-81 (2005), which is hereby incorporated by reference in itsentirety. In Table 2 and throughout the specification, X^(s15), X^(s21),X^(s22), X^(s23) and X^(s27) each independently refer to nonfunctionalamino acid residues.

TABLE 3 HmK, BgK, AeK and AsKS peptide and peptide analog sequencesShort- hand SEQ desig- ID Sequence/structure nation NO:RTCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC HmK 6ATCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC HmK-A1 201STCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC HmK-S1 202 TCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC HmK-d1 203 SCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC HmK- 204 d1/S2 TCIDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC HmK- 205 d1/I4 TCKDTIPVSECTDIRCRTSMKYRLNLCRKTCGSC HmK- 206 d1/T6 TCKDLIPKSECTDIRCRTSMKYRLNLCRKTCGSC HmK- 207 d1/K9 TCKDLIPVSRCTDIRCRTSMKYRLNLCRKTCGSC HmK- 208 d1/R11 TCKDLIPVSECTAIRCRTSMKYRLNLCRKTCGSC HmK- 209 d1/A14 TCKDLIPVSECTDFRCRTSMKYRLNLCRKTCGSC HmK- 210 d1/F15 TCKDLIPVSECTDIQCRTSMKYRLNLCRKTCGSC HmK- 211 d1/Q16 TCKDLIPVSECTDIRCKTSMKYRLNLCRKTCGSC HmK- 212 d1/K18 TCKDLIPVSECTDIRCRHSMKYRLNLCRKTCGSC HmK- 213 d1/H19 TCKDLIPVSECTDIRCRTSMKYRLSLCRKTCGSC HmK- 214 d1/S26 TCKDLIPVSECTDIRCRTSMKYRLNFCRKTCGSC HmK- 215 d1/F27 TCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGTC HmK- 216 d1/T34 TCKDLIPVSRCTDIRCRTSMKYRLNFCRKTCGSC HmK- 217 d1/R11/ F27ATCKDLIPVSRCTDIRCRTSMKYRLNFCRKTCGSC HmK- 218 A1/R11/ F27 TCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGSC HmK- 219 d1/Z1 TCIDTIPKSRCTAFQCRTSMKYRLNFCRKTCGSC HmK- 220 d1/Z2 TCADLIPASRCTAIACRTSMKYRLNFCRKTCGSC HmK- 221 d1/Z3 TCADLIPASRCTAIACKHSMKYRLNFCRKTCGSC HmK- 222 d1/Z4 TCADLIPASRCTAIACAHSMKYRLNFCRKTCGSC HmK- 223 d1/Z5RTCKDLIPVSECTDIRCRTSMX^(h22)YRLNLCRKTCGSC HmK-X22 224ATCKDLX^(h6)PVSRCTDIRCRTSMKX^(h22)RLNX^(h26)CR HmK-X6, 225 KTCGSC 22, 26VCRDWFKETACRHAKSLGNCRTSQKYRANCAKTCELC BgK 9ACRDWFKETACRHAKSLGNCRTSQKYRANCAKTCELC BgK-A1 226VCADWFKETACRHAKSLGNCRTSQKYRANCAKTCELC BgK-A3 227VCRDAFKETACRHAKSLGNCRTSQKYRANCAKTCELC BgK-A5 228VCRDWFKATACRHAKSLGNCRTSQKYRANCAKTCELC BgK-A8 229VCRDWFKEAACRHAKSLGNCRTSQKYRANCAKTCELC BgK-A9 230VCRDWFKETACAHAKSLGNCRTSQKYRANCAKTCELC BgK-A12 231VCRDWFKETACRHAASLGNCRTSQKYRANCAKTCELC BgK-A15 232VCRDWFKETACRHAKALGNCRTSQKYRANCAKTCELC BgK-A16 233VCRDWFKETACRHAKSAGNCRTSQKYRANCAKTCELC BgK-A17 234VCRDWFKETACRHAKSLGNCATSQKYRANCAKTCELC BgK-A21 235VCRDWFKETACRHAKSLGNCRASQKYRANCAKTCELC BgK-A22 236VCRDWFKETACRHAKSLGNCRTSQKYAANCAKTCELC BgK-A27 237VCRDWFKETACRHAKSLGNCRTSQKYRANCAATCELC BgK-A32 238VCRDWFKETACRHAKSLGNCRTSQKYRANCAKACELC BgK-A33 239VCRDWFKETACRHAKSLGNCRTSQKYRANCAKTCALC BgK-A35 240VCRDWFKETACRHAKSLGNCRTSQKYRANCAKTCEAC BgK-A37 241GCKDNFSANTCKHVKANNNCGSQKYATNCAKTCGKC AeK 7ACKDNFAAATCKHVKENKNCGSQKYATNCAKTCGKC AsKS 8

In Table 3 and throughout the specification, X^(h6), X^(h22), X^(h26)are each independently nonfunctional residues.

TABLE 4 MgTx peptide and MgTx peptide analog sequences Short- hand SEQdesig- ID Sequence/structure nation NO:TIINVKCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH MgTx 28TIINVACTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH MgTx- 242 A6TIINVSCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH MgTx- 243 S6TIINVKCTSPAQCLPPCKAQFGQSAGAKCMNGKCKCYPH MgTx- 244 A11TIINVKCTSPKQCLPPCAAQFGQSAGAKCMNGKCKCYPH MgTx- 245 A18TIINVKCTSPKQCLPPCKAQFGQSAGAACMNGKCKCYPH MgTx- 246 A28TIINVKCTSPKQCLPPCKAQFGQSAGAKCMNGACKCYPH MgTx- 247 A33TIINVKCTSPKQCLPPCKAQFGQSAGAKCMNGKCACYPH MgTx- 248 A35TIINVKCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPN MgTx- 249 H39NTIINVACTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPN MgTx- 250 A6/ H39NTIINVSCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYS MgTx- 251 S6/ 38/d39TIITISCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH MgTx- 252 T4/I5/ S6   TISCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH MgTx- 253 d3/T4/ I5/S6   TISCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCFGR MgTx- 254 Pi2   NVACTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH MgTx- 255 d3/A6QFTNVSCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYS MgTx- 256 ChTxQFTDVDCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYQ MgTx- 257 IbTx IINVSCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH MgTx- 258 Z1 IITISCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH MgTx- 259 Z2GVIINVSCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCY MgTx- 260 PH Z3

Many peptides as described in Table 4 can be prepared as described in WO95/03065, published Feb. 2, 1995, for which the applicant is Merck &Co., Inc. That application corresponds to U.S. Ser. No. 07/096,942,filed 22 Jul. 1993, which is hereby incorporated by reference in itsentirety.

TABLE 5 AgTx2 peptide and AgTx2 peptide analog sequences Short- hand SEQdesig- ID Sequence/structure nation NO:GVPINVSCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPK AgTx2 23GVPIAVSCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPK AgTx2- 261 A5GVPINVSCTGSPQCIAPCKDAGMRFGKCMNRKCHCTPK AgTx2- 262 A16GVPINVSCTGSPQCIKPCADAGMRFGKCMNRKCHCTPK AgTx2- 263 A19GVPINVSCTGSPQCIKPCKDAGMAFGKCMNRKCHCTPK AgTx2- 264 A24GVPINVSCTGSPQCIKPCKDAGMRFGACMNRKCHCTPK AgTx2- 265 A27GVPINVSCTGSPQCIKPCKDAGMRFGKCMNAKCHCTPK AgTx2- 266 A31GVPINVSCTGSPQCIKPCKDAGMRFGKCMNRACHCTPK AgTx2- 267 A32GVPINVSCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPA AgTx2- 268 A38GVPIAVSCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPA AgTx2- 269 A5/A38GVPINVSCTGSPQCIKPCKDAGMRFGKCMNGKCHCTPK AgTx2- 270 G31GVPIIVSCKGSRQCIKPCKDAGMRFGKCMNGKCHCTPK AgTx2- 271 OSK_z1GVPIIVSCKISRQCIKPCKDAGMRFGKCMNGKCHCTPK AgTx2- 272 OSK_z2GVPIIVKCKGSRQCIKPCKDAGMRFGKCMNGKCHCTPK AgTx2- 273 OSK_z3GVPIIVKCKISRQCIKPCKDAGMRFGKCMNGKCHCTPK AgTx2- 274 OSK_z4GVPIIVKCKISRQCIKPCKDAGMRFGKCMNGKCHCTPK AgTx2- 275 OSK_z5

TABLE 6 Heteromitrus spinnifer (HsTx1) peptide and HsTx1peptide analog sequences SEQ Short-hand ID Sequence/structuredesignation NO: ASCRTPKDCADPCRKETGCPYGKCMNRKCKCNRC HsTx1 61ASCXTPKDCADPCRKETGCPYGKCMNRKCKCNRC HsTx1-X4 276ASCATPKDCADPCRKETGCPYGKCMNRKCKCNRC HsTx1-A4 277ASCRTPXDCADPCRKETGCPYGKCMNRKCKCNRC HsTx1-X7 278ASCRTPADCADPCRKETGCPYGKCMNRKCKCNRC HsTx1-A7 279ASCRTPKDCADPCXKETGCPYGKCMNRKCKCNRC HsTx1-X14 280ASCRTPKDCADPCAKETGCPYGKCMNRKCKCNRC HsTx1-A14 281ASCRTPKDCADPCRXETGCPYGKCMNRKCKCNRC HsTx1-X15 282ASCRTPKDCADPCRAETGCPYGKCMNRKCKCNRC HsTx1-A15 283ASCRTPKDCADPCRKETGCPYGXCMNRKCKCNRC HsTx1-X23 284ASCRTPKDCADPCRKETGCPYGACMNRKCKCNRC HsTx1-A23 285ASCRTPKDCADPCRKETGCPYGKCMNXKCKCNRC HsTx1-X27 286ASCRTPKDCADPCRKETGCPYGKCMNAKCKCNRC HsTx1-A27 287ASCRTPKDCADPCRKETGCPYGKCMNRXCKCNRC HsTx1-X28 288ASCRTPKDCADPCRKETGCPYGKCMNRACKCNRC HsTx1-A28 289ASCRTPKDCADPCRKETGCPYGKCMNRKCXCNRC HsTx1-X30 290ASCRTPKDCADPCRKETGCPYGKCMNRKCACNRC HsTx1-A30 291ASCRTPKDCADPCRKETGCPYGKCMNRKCKCNXC HsTx1-X33 292ASCRTPKDCADPCRKETGCPYGKCMNRKCKCNAC HsTx1-A33 293

Peptides as described in Table 5 can be prepared as described in U.S.Pat. No. 6,689,749, issued Feb. 10, 2004 to Lebrun et al., which ishereby incorporated by reference in its entirety.

TABLE 7Orthochirus scrobiculosus (OSK1) peptide and OSK1 peptide analog sequencesSEQ Short-hand ID Sequence/structure designation NO:GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK OSK1 25GVIINVSCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK OSK1-S7 1303GVIINVKCKISRQCLKPCKKAGMRFGKCMNGKCHCTPK OSK1-K16 294GVIINVKCKISRQCLEPCKDAGMRFGKCMNGKCHCTPK OSK1-D20 295GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK OSK1-K16, D20 296GVIINVSCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK OSK1-S7, K16, D20 1308GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK OSK1-P12, K16, D20 297GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYPK OSK1-K16, D20, Y36 298Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK Ac-OSK1-P12, 562 K16, D20GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ OSK1-P12, K16, 563 D20-NH₂Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-OSK1-P12, 564K16, D20-NH₂ GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYPK-NH₂ OSK1-K16, D20,565 Y36-NH₂ Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYPK Ac-OSK1-K16, 566D20, Y36 Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYPK-NH₂ Ac-OSK1-K16, D20,567 Y36-NH₂ GVIINVKCKISRQCLKPCKKAGMRFGKCMNGKCHCTPK-NH₂ OSK1-K16-NH₂ 568Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCMNGKCHCTPK Ac-OSK1-K16 569Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCMNGKCHCTPK-NH₂ Ac-OSK1-K16-NH₂ 570Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCMNGKCHCTPK Ac-OSK1-D20 571GVIINVKCKISRQCLEPCKDAGMRFGKCMNGKCHCTPK-NH₂ OSK1-D20-NH₂ 572Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-OSK1-D20-NH₂ 573GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH₂ OSK1-NH₂ 574Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK Ac-OSK1 575Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH₂ Ac-OSK1-NH₂ 576GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ OSK1-K16, D20-NH₂ 577Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK Ac-OSK1-K16, 578 D20Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-OSK1-K16, D20- 579 NH₂VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK Δ1-OSK1 580Ac-VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK Ac-Δ1-OSK1 581VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH₂ Δ1-OSK1-NH₂ 582Ac-VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH₂ Ac-Δ1-OSK1-NH₂ 583GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK OSK1-A34 584Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK Ac-OSK1-A34 585GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-NH₂ OSK1-A34-NH₂ 586Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-NH₂ Ac-OSK1-A34-NH₂ 587VIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK Δ1-OSK1-K16, D20 588Ac-VIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK Ac-Δ1-OSK1-K16, 589 D20VIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Δ1-OSK1-K16, D20- 590 NH₂Ac-VIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-Δ1-OSK1-K16, 591 D20-NH₂NVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK (Δ1-4)-OSK1-K16, 592 D20Ac-NVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK Ac-(Δ1-4)-OSK1- 593 K16, D20NVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ (Δ1-4)-OSK1-K16, 594 D20-NH₂Ac-NVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-(Δ1-4)-OSK1- 595K16, D20-NH₂ KCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK (Δ1-6)-OSK1-K16, 596 D20Ac-KCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK Ac-(Δ1-6)-OSK1- 597 K16, D20KCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ (Δ1-6)-OSK1-K16, 598 D20-NH₂Ac-KCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-(Δ1-6)-OSK1- 599 K16, D20-NH₂CKISRQCLKPCKDAGMRFGKCMNGKCHCTPK (Δ1-7)-OSK1-K16, 600 D20Ac-CKISRQCLKPCKDAGMRFGKCMNGKCHCTPK Ac-(Δ1-7)-OSK1- 601 K16, D20CKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ (Δ1-7)-OSK1-K16, 602 D20-NH₂Ac-CKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-(Δ1-7)-OSK1- 603 K16, D20-NH₂GVIINVKCKISRQCLKPCKDAGMRNGKCMNGKCHCTPK OSK1-K16, D20, 604 N25GVIINVKCKISRQCLKPCKDAGMRNGKCMNGKCHCTPK-NH₂ OSK1-K16, D20, 605 N25-NH₂Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCMNGKCHCTPK Ac-OSK1-K16, 606 D20, N25Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCMNGKCHCTPK-NH₂ Ac-OSK1-K16, D20, 607N25-NH₂ GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK OSK1-K16, D20, 608 R31GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-NH₂ OSK1-K16, D20, 609 R31-NH₂Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK Ac-OSK1-K16, 610 D20, R31Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-NH₂ Ac-OSK1-K16, D20, 611R31-NH₂ GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK OSK1-K12, K16, 612R19, D20 Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK Ac-OSK1-K12, K16, 613R19, D20 GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK-NH₂ OSK1-K12, K16, 614R19, D20-NH₂ Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK-NH₂Ac-OSK1-K12, K16, 615 R19, D20-NH₂ TIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPKΔ1-OSK1-T2, K16, 616 D20 Ac-TIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPKAc-Δ1-OSK1-T2, 617 K16, D20 TIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂Δ1-OSK1-T2, K16, 618 D20-NH₂Ac-TIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-Δ1-OSK1-T2, 619K16, D20-NH₂ GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK OSK1-K3 620Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK Ac-OSK1-K3 621GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH₂ OSK1-K3-NH₂ 622Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH₂ Ac-OSK1-K3-NH₂ 623GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK OSK1-K3, A34 624GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK OSK1-K3, K16, D20 625GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCACTPK OSK1-K3, K16, D20, 626 A34Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK Ac-OSK1-K3, A34 627GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-NH₂ OSK1-K3, A34-NH₂ 628Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-NH₂ Ac-OSK1-K3, A34- 629 NH₂Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCACTPK Ac-OSK1-K3, K16, 630 D20, A34GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCACTPK-NH₂ OSK1-K3, K16, D20, 631A34-NH₂ Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCACTPK-NH₂ Ac-OSK1-K3, K16,632 D20, A34-NH₂ Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPKAc-OSK1-K3, K16, 633 D20 GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂OSK1-K3, K16, D20- 634 NH₂ Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂Ac-OSK1-K3, K16, 635 D20-NH₂ GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTΔ36-38-OSK1-K16, 636 D20 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCTPKOSK1-O16, D20 980 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCTPK OSK1-hLys981 16, D20 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCTPK OSK1-hArg 98216, D20 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCTPK OSK1-Cit 16, D20 983GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK OSK1-hCit 984 16, D20GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCTPK OSK1-Dpr 16, D20 985GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCHCTPK OSK1-Dab 16, D20 986GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCYPK OSK1-O16, D20, Y36 987GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCYPK OSK1-hLys 988 16, D20, Y36GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCYPK OSK1-hArg 989 16, D20, Y36GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCYPK OSK1-Cit 990 16, D20, Y36GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYPK OSK1-hCit 991 16, D20, Y36GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCYPK OSK1-Dpr 992 16, D20, Y36GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCHCYPK OSK1-Dab 993 16, D20, Y36GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYPK OSK1- 994 K16, D20, A34, Y36GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCGCYPK OSK1- 995 K16, D20, G34, Y36GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACFPK OSK1- 996 K16, D20, A34, F36GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACWPK OSK1- 997 K16, D20, A34, W36GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACYPK OSK1- 998 K16, E20, A34, Y36GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACTPK OSK1-O16, D20, A34 999GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACTPK OSK1-hLys 1000 16, D20, A34GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACTPK OSK1-hArg 1001 16, D20, A34GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACTPK OSK1-Cit 1002 16, D20, A34GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK OSK1-hCit 1003 16, D20, A34GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACTPK OSK1-Dpr 1004 16, D20, A34GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACTPK OSK1-Dab 1005 16, D20, A34GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHC Δ36-38, OSK1- 1006 O16, D20,GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHC Δ36-38, OSK1- 1007 hLys 16, D20GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHC Δ36-38, OSK1- 1008 hArg 16, D20GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHC Δ36-38, OSK1-Cit 1009 16, D20GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHC Δ36-38, OSK1- 1010 hCit 16, D20GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHC Δ36-38, OSK1-Dpr 1011 16, D20GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCHC Δ36-38, OSK1- 1012 Dab16, D20GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCAC Δ36-38, OSK1- 1013 O16, D20, A34GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCAC Δ36-38, OSK1- 1014hLys 16, D20, A34 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCAC Δ36-38, OSK1-1015 hArg 16, D20, A34 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACΔ36-38, OSK1-Cit 1016 16, D20, A34GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHC Δ36-38, OSK1- 1017hCit 16, D20, A34 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACΔ36-38, OSK1-Dpr 1018 16, D20, A34GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCAC Δ36-38, OSK1-Dab 101916, D20, A34 GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCGCYGG OSK1- 1020K16, D20, G34, Y36, G37, G38 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCYGGOSK1- 1021 O16, D20, Y36, G37, G38GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCYGG OSK1-hLys 102216, D20, Y36, G37, G38 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCYGGOSK1-hArg 1023 16, D20, Y36, G37, G38GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCYGG OSK1-Cit 102416, D20, Y36, G37, G38 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYGGOSK1-hCit 1025 16, D20, Y36, G37, G38GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCYGG OSK1-Dpr 102616, D20, Y36, G37, G38 GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYGG OSK1- 1027K16, D20, A34, Y36, G37, G38 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACYGGOSK1- 1028 O16, D20, A34, Y36, G37, G38GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACYGG OSK1-hLys 102916, D20, A34, Y36, G37, G38 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACYGGOSK1-hArg 1030 16, D20, A34, Y36, G37, G38GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACYGG OSK1-Cit 103116, D20, A34, Y36, G37, G38 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYGGOSK1-hCit 1032 16, D20, A34, Y3, G37, G38GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACYGG OSK1-Dpr 103316, D20, A34, Y36, G37, G38 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACYGGOSK1-Dab 1034 16, D20, A34, Y36, G37, G38GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYG Δ38, OSK1- 1035K16, D20, A34, Y36, G37 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCGGG OSK1-1036 O16, D20, G36-38 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCGGGOSK1-hLys 1037 16, D20, G36-38GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCGGG OSK1-hArg 103816, D20, G36-38 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCGGG OSK1-Cit 103916, D20, G36-38 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCGGG OSK1-hCit1040 16, D20, G36-38 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCGGG OSK1-Dpr1041 16, D20, G36-38 GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACFGG OSK1- 1042K16, D20, A34, F36, G37, G38 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACGGGOSK1- 1043 O16, D20, A34, G36- 38GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACGGG OSK1-hLys 104416, D20, A34, G36-38 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACGGGOSK1-hArg 1045 16, D20, A34, G36-38GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACGGG OSK1-Cit 104616, D20, A34, G36-38 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCACGGGOSK1-hCit 1047 16, D20, A34, G36-38GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACGGG OSK1-Dpr 104816, D20, A34, G36-38 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACGGG OSK1-Dab1049 16, D20, A34, G36-38 GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACGGΔ38, OSK1- 1050 K16, D20, A34, G36- 37GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYG Δ38, OSK1- 1051K16, D20, A35, Y36, G37 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACGG Δ38, OSK1-1052 O16, D20, A35, Y36, G37 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCTPKOSK1-hLys 1053 16, E20 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCTPKOSK1-hArg 1054 16, E20 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCTPKOSK1-Cit 16, E20 1055 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCTPKOSK1-hCit 1056 16, E20 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCTPKOSK1-Dpr 16, E20 1057 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCTPKOSK1-Dab 16, E20 1058 GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCYPKOSK1-O16, E20, Y36 1059 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCYPKOSK1-hLys 1060 16, E20, Y36 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCYPKOSK1-hArg 1061 16, E20, Y36 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCYPKOSK1-Cit 1062 16, E20, Y36 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYPKOSK1-hCit 1063 16, E20, Y36 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCYPKOSK1-Dpr 1064 16, E20, Y36 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCYPKOSK1-Dab 1065 16, E20, Y36 GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACTPKOSK1-O16, E20, A34 1066 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACTPKOSK1-hLys 1067 16, E20, A34 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACTPKOSK1-hArg 1068 16, E20, A34 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACTPKOSK1-Cit 1069 16, E20, A34 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCTPKOSK1-hCit 1070 16, E20, A34 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACTPKOSK1-Dpr 1071 16, E20, A34 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACTPKOSK1-Dab 1072 16, E20, A34 GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCΔ36-38, OSK1- 1073 O16, E20, GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCΔ36-38, OSK1- 1074 hLys 16, E20 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCΔ36-38, OSK1- 1075 hArg 16, E20 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCΔ36-38, OSK1-Cit 1076 16, E20 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCΔ36-38, OSK1- 1077 hCit16, E20 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCΔ36-38, OSK1-Dpr 1078 16, E20 GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACΔ36-38, OSK1- 1079 O16, E20, A34GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCAC Δ36-38, OSK1- 1080hLys 16, E20, A34 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCAC Δ36-38, OSK1-1081 hArg 16, E20, A34 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACΔ36-38, OSK1-Cit 1082 16, E20, A34GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHC Δ36-38, OSK1- 1083hCit 16, E20, A34 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACΔ36-38, OSK1-Dpr 1084 16, E20, A34GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCAC Δ36-38, OSK1-Dab 108516, E20, A34 GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCHCYGG OSK1- 1086K16, E20, Y36, G37, G38 GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCYGG OSK1-1087 O16, E20, Y36, G37, G38 GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCHCYGΔ38 OSK1- 1088 K16, E20, Y36, G37 GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACYGΔ38 OSK1- 1089 K16, E20, A34, Y36, G37GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCYGG OSK1-hLys 109016, E20, Y36, G37, G38 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCYGGOSK1-hArg 1091 16, E20, Y36, G37, G38GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCYGG OSK1-Cit 109216, E20, Y36, G37, G38 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYGGΔ37-38, OSK1- 1093 hCit 16, E20, Y36, G37, G38GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCYGG OSK1-Dpr 109416, E20, Y36, G37, G38 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCYGGOSK1-Dab 1095 16, E20, Y36, G37, G38GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACYG Δ38, OSK1- 1096K16, E20, A34, Y36, G37 GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACYGG OSK1-1097 O16, E20, A34, Y36, G37, G38GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACYGG OSK1-hLys 109816, E20, A34, Y36, G37, G38 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACYGGOSK1-hArg 1099 16, E20, A34, Y36, G37, G38GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACYGG OSK1-Cit 110016, E20, A34, Y36, G37, G38 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYGGOSK1-hCit 1101 16, E20, A34, Y3, G37, G38GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACYGG OSK1-Dpr 110216, E20, A34, Y36, G37, G38 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACYGGOSK1-Dab 1103 16, E20, A34, Y36, G37, G38GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACFGG OSK1- 1104K16, D20, A34, F36, G37, G38 GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCGGGOSK1- 1105 O16, E20, G36-38 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCGGGOSK1-hLys 1106 16, E20, G36-38GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCGGG OSK1-hArg 110716, E20, G36-38 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCGGG OSK1-Cit 110816, E20, G36-38 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCGGG OSK1-hCit1109 16, E20, G36-38 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCGGG OSK1-Dpr1110 16, E20, G36-38 GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACGGG OSK1- 1111O16, E20, A34, G36- 38 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACGGGOSK1-hLys 1112 16, E20, A34, G36-38GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACGGG OSK1-hArg 111316, E20, A34, G36-38 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACGGG OSK1-Cit1114 16, E20, A34, G36-38 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCACTPOSK1-hCit 1115 16, E20, A34, G36-38GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACTP OSK1-Dpr 111616, E20, A34, G36-38 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACTP OSK1-Dab1117 16, E20, A34, G36-38 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCTPK-NH2OSK1-O16, D20- 1118 amide GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCTPK-OSK1-hLys 1119 NH2 16, D20-amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCTPK- OSK1-hArg 1120 NH216, D20-amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCTPK-NH2 OSK1-Cit1121 16, D20-amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK-OSK1-hCit 1122 NH2 16, D20-amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCTPK-NH2 OSK1-Dpr 112316, D20-amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCHCTPK-NH2OSK1-Dab 16, D20 1124 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCYPK-NH2 OSK1-1125 O16, D20, Y36- amide GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCYPK-OSK1-hLys 1126 NH2 16, D20, Y36- amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCYPK- OSK1-hArg 1127 NH216, D20, Y36- amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCYPK-NH2OSK1-Cit 1128 16, D20, Y36- amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYPK- OSK1-hCit 1129 NH216, D20, Y36- amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCYPK-NH2OSK1-Dpr 1130 16, D20, Y36- amideGVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCHCYPK-NH2 OSK1-Dab 113116, D20, Y36- amide GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACTPK-NH2 OSK1-1132 O16, D20, A34- amide GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACTPK-OSK1-hLys 1133 NH2 16, D20, A34- amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACTPK- OSK1-hArg 1134 NH216, D20, A34- amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACTPK-NH2OSK1-Cit 1135 16, D20, A34- amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCACTPK- OSK1-hCit 1136 NH216, D20, A34- amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACTPK-NH2OSK1-Dpr 1137 16, D20, A34- amideGVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACTPK-NH2 OSK1-Dab 113816, D20, A34- amide GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHC-NH2Δ36-38, OSK1- 1139 O16, D20, - amideGVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHC-NH2 Δ36-38, OSK1- 1140hLys 16, D20- amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHC-NH2Δ36-38, OSK1- 1141 hArg 16, D20- amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHC-NH2 Δ36-38, OSK1-Cit 114216, D20-amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHC-NH2 Δ36-38, OSK1-1143 hCit16, D20- amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHC-NH2Δ36-38, OSK1-Dpr 1144 16, D20-amideGVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCAC-NH2 Δ36-38, OSK1- 1145O16, D20, A34- amide GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCAC-NH2Δ36-38, OSK1- 1146 hLys 16, D20, A34- amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCAC-NH2 Δ36-38, OSK1- 1147hArg 16, D20, A34- amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCAC-NH2Δ36-38, OSK1-Cit 1148 16, D20, A34- amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHC-NH2 Δ36-38, OSK1- 1149hCit16, D20, A34 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCAC-NH2Δ36-38, OSK1-Dpr 1150 16, D20, A34- amideGVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCAC-NH2 Δ36-38, OSK1-Dab 115116, D20, A34- amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYGG-NH2 OSK1-1152 O16, D20, Y36, G37, G38- amideGVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCYGG-NH2 OSK1- 1153O16, D20, Y36, G37, G38 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCYGG-OSK1-hLys 1154 NH2 16, D20, Y36, G37, G38- amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCYGG- OSK1-hArg 1155 NH216, D20, Y36, G37, G38- amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCYGG-NH2 OSK1-Cit 115616, D20, Y36, G37, G38- amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYGG- OSK1- 1157 NH2hCit16, D20, Y36, G37, G38-amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCYGG-NH2 OSK1-Dpr 115816, D20, Y36, G37, G38- amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCFGG-NH2OSK1- 1159 K16, D20, F36, G37, G38- amideGVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYG-NH2 Δ38-OSK1- 1160K16, D20, Y36, G37- amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYG-NH2Δ38-OSK1- 1161 K16, D20, A34, Y36, G37-amideGVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACYGG-NH2 OSK1- 1162O16, D20, A34, Y36, G37, G38-amideGVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACYGG- OSK1-hLys 1163 NH216, D20, A34, Y36, G37, G38-amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACYGG- OSK1-hArg 1164 NH216, D20, A34, Y36, G37, G38-amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACYGG-NH2 OSK1-Cit 116516, D20, A34, Y36, G37, G38 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCACYGG-OSK1- 1166 NH2 hCit16, D20, A34, Y3, G37, G38-amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACYGG-NH2 OSK1-Dpr 116716, D20, A34, Y36, G37, G38-amideGVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACYGG-NH2 OSK1-Dab 116816, D20, A34, Y36, G37, G38-amideGVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYGG-NH2 OSK1- 1169K16, D20, A34, Y36, G37, G38-amideGVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCGGG-NH2 OSK1- 1170 O16, D20, G36-38-amide GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCGGG- OSK1-hLys 1171 NH216, D20, G36-38- amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCGGG-OSK1-hArg 1172 NH2 16, D20, G36-38- amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCGGG-NH2 OSK1-Cit 117316, D20, G36-38- amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCGGG-OSK1- 1174 NH2 hCit16, D20, G36- 38-amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCGGG-NH2 OSK1-Dpr 117516, D20, G36-38- amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACGGG-NH2 OSK1-1176 K16, D20, A34, G36- 38-amideGVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACFGG-NH2 OSK1- 1177O16, D20, A34, F36, G37-38-amideGVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACGGG-NH2 OSK1- 1178O16, D20, A34, G36- 38-amideGVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACGGG- OSK1-hLys 1179 NH216, D20, A34, G36- 38-amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACGGG-OSK1-hArg 1180 NH2 16, D20, A34, G36- 38-amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACGGG-NH2 OSK1-Cit 118116, D20, A34, G36- 38-amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCACGGG-OSK1- 1182 NH2 hCit16, D20, A34, G36- 38-amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACGGG-NH2 OSK1-Dpr 118316, D20, A34, G36- 38-amideGVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACGGG-NH2 OSK1-Dab 118416, D20, A34, G36- 38-amide GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCTPK-NH2OSK1-O16, E20- 1185 amide GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCTPK-OSK1-hLys 1186 NH2 16, E20-amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCTPK- OSK1-hArg 1187 NH216, E20-amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCTPK-NH2 OSK1-Cit1188 16, E20-amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK- OSK1-1189 NH2 hCit16, E20- amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCTPK-NH2 OSK1-Dpr 119016, E20-amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCTPK-NH2 OSK1-Dab1191 16, E20-amide GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCYPK-NH2 OSK1- 1192O16, E20, Y36- amide GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCYPK-OSK1-hLys 1193 NH2 16, E20, Y36- amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCYPK- OSK1-hArg 1194 NH216, E20, Y36- amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCYPK-NH2OSK1-Cit 1195 16, E20, Y36- amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYPK- OSK1- 1196 NH2hCit16, E20, Y36- amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCYPK-NH2OSK1-Dpr 1197 16, E20, Y36- amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCYPK-NH2 OSK1-Dab 119816, E20, Y36- amide GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACTPK-NH2 OSK1-1199 O16, E20, A34- amide GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACTPK-OSK1-hLys 1200 NH2 16, E20, A34- amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACTPK- OSK1-hArg 1201 NH216, E20, A34- amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACTPK-NH2OSK1-Cit 1202 16, E20, A34- amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCACTPK- OSK1- 1203 NH2hCit16, E20, A34- amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACTPK-NH2OSK1-Dpr 1204 16, E20, A34- amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACTPK-NH2 OSK1-Dab 120516, E20, A34- amide GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHC-NH2Δ36-38, OSK1- 1206 O16, E20, - amideGVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHC-NH2 Δ36-38, OSK1- 1207hLys 16, E20- amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHC-NH2Δ36-38, OSK1- 1208 hArg 16, E20- amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHC-NH2 Δ36-38, OSK1-Cit 120916, E20-amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHC-NH2 Δ36-38, OSK1-1210 hCit16, E20- amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHC-NH2Δ36-38, OSK1-Dpr 1211 16, E20-amideGVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCAC-NH2 Δ36-38, OSK1- 1212O16, E20, A34- amide GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCAC-NH2Δ36-38, OSK1- 1213 hLys16, E20, A34- amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCAC-NH2 Δ36-38, OSK1- 1214hArg16, E20, A34- amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCAC-NH2Δ36-38, OSK1-Cit 1215 16, E20, A34- amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHC-NH2 Δ36-38, OSK1- 1216hCit16, E20, A34- amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCAC-NH2Δ36-38, OSK1-Dpr 1217 16, E20, A34- amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCAC-NH2 Δ36-38, OSK1-Dab 121816, E20, A34- amide GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCHCWGG-NH2 OSK1-1219 O16, E20, W36, G37, G38-amideGVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCYGG-NH2 OSK1- 1220O16, E20, Y36, G37, G38- amideGVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCYGG- OSK1-hLys 1221 NH216, E20, Y36, G37, G38- amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCYGG- OSK1-hArg 1222 NH216, E20, Y36, G37, G38- amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCYGG-NH2 OSK1-Cit 122316, E20, Y36, G37, G38- amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG- OSK1-hCit 1224 NH216, E20, Y36, G37, G38- amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCYGG-NH2 OSK1-Dpr 122516, E20, Y36, G37, G38- amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCYGG-NH2 OSK1-Dpr 122616, E20, Y36, G37, G38- amide GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACYGG-NH2OSK1- 1227 K16, E20, A34, Y36, G37, G38-amideGVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACYGG-NH2 OSK1- 1228O16, E20, A34, Y36, G37, G38-amideGVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACYGG- OSK1-hLys 1229 NH216, E20, A34, Y36, G37, G38-amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACYGG- OSK1-hArg 1230 NH216, E20, A34, Y36, G37, G38-amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACYGG-NH2 OSK1-Cit 123116, E20, A34, Y36, G37, G38-amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG- OSK1-hCit 1232 NH216, E20, A34, Y3, G37, G38-amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACYGG-NH2 OSK1-Dpr 123316, E20, A34, Y36, G37, G38-amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACYGG-NH2 OSK1-Dab 123416, E20, A34, Y36, G37, G38-amideGVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCGGG-NH2 OSK1- 1235 O16, E20, G36-38-amide GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCGGG- OSK1-hLys 1236 NH216, E20, G36-38- amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCGGG-OSK1-hArg 1237 NH2 16, E20, G36-38- amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCGGG-NH2 OSK1-Cit 123816, E20, G36-38- amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCGGG-OSK1-hCit 1239 NH2 16, E20, G36-38- amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCGGG-NH2 OSK1-Dpr 124016, E20, G36-38- amide GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACGGG-NH2 OSK1-1241 O16, E20, A34, G36- 38-amideGVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACGGG- OSK1-hLys 1242 NH216, E20, A34, G36- 38-amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACGGG-OSK1-hArg 1243 NH2 16, E20, A34, G36- 38-amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACGGG-NH2 OSK1-Cit 124416, E20, A34, G36- 38-amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCACTP-NH2 Δ38 OSK1-hCit 124516, E20, A34- amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACGGG-NH2OSK1-Dpr 1246 16, E20, A34, G36- 38-amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACGGG-NH2 OSK1-Dab 124716, E20, A34, G36- 38-amideGVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMNGKCACYGG-NH2 OSK1-K 124816, CPA20, A34, Y36, G37, G38-amideGVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMNGKCACGGG-NH2 OSK1-K 124916, CPA20, A34, G36- 38-amideGVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMNGKCACY-NH2 Δ37-38OSK1-K 125016, CPA20, A34, Y36- amide Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYGG-NH2Acetyl-OSK1-K 1251 16, D20, A34, Y36, G37, G38-amideGVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMNGKCACYGG-NH2 OSK1-K 16, 1252Aad20, A34, Y36, G37, G38-amideGVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMNGKCHCYGG-NH2 OSK1-K 16, 1253Aad20, Y36, G37, G38- amide GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMNGKCACYGGOSK1-K 16, 1254 Aad20, A34, Y36, G37, G38GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCACYGG-NH2 OSK1-H 125516, D20, A34, Y36, G37, G38-amide GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCACYGGOSK1-H 1256 16, D20, A34, Y36, G37, G38GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCACY-NH2 Δ37-38-OSK1-H 125716, D20, A34, Y36- amide GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCHCYGG-NH2OSK1-H 1258 16, D20, A34, Y36, G37, G38-amideGVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCHCYGG OSK1-H 125916, D20, A34, Y36, G37, G38 GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCHCYPKOSK1-H 1260 16, D20, A34, Y36, GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCACΔ36-38 OSK1-H 1261 16, D20, A34, Y36,GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[1Nal]GG- OSK1-K 1262 NH216, D20, A34, 1Nal36, G37, G38-amideGVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[1Nal]PK- OSK1-K 1263 NH216, D20, A34, 1Nal36- amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[2Nal]GG-OSK1-K 1264 NH2 16, D20, A34, 2Nal36, G37, G38-amideGVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[Cha]GG-NH2 OSK1-K 126516, D20, A34, Cha36, G37, G38-amideGVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[MePhe]GG- OSK1-K 1266 NH216, D20, A34, MePhe36, G37, G38- amideGVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[BiPhA]GG- OSK1-K 1267 NH216, D20, A34, BiPhA36, G37, G38- amideGVIINVKCKISRQCLKPCKDAGMRFGKCMNGKC[Aib]CYGG-NH2 OSK1-K 16, D20, 1268Aib34, Y36, G37, G38- amideGVIINVKCKISRQCLKPCKDAGMRFGKCMNGKC[Abu]CYGG-NH2 OSK1-K 16, D20, 1269Abu34, Y36, G37, G38- amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[1Nal]Δ37-38 OSK1-H 1270 16, D20, A34, 1Nal36, - amideGVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCAC[1Nal]GG- OSK1-H 1271 NH216, D20, A34, 1Nal36, G37, G38- amideGVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[4Bip]-NH2 Δ37-38 OSK1-H 127216, D20, A34, 4Bip 36, -amideGVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCAC[4Bip]GG- OSK1-H 1273 NH216, D20, A34, 4Bip 36, G37, G38- amideGVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCGGG OSK1-K16, E20, G36- 1274 38

TABLE 7A Additional useful OSK1 peptide analog sequences SEQ ID NOSequence 1391 GVIINVKCKISAQCLKPCRDAGMRFGKCMNGKCACTPK 1392GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1393GVIINVKCKISPQCLKPCKDAGIRFGKCINGKCACTPK 1394GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACTPK 1395GGGGSGVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1396GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHC 1397GVIINVKCKISPQCLOPCKEAGMRFGKCMNGKCHCTY[Nle] 1398GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTY[Nle] 1399GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCGGG 1400AVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1401GAIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1402GVAINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1403GVIANVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1404GVIIAVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1405GVIINAKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1406GVIINVACKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1407GVIINVKCAISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1408GVIINVKCKASRQCLEPCKKAGMRFGKCMNGKCHCTPK 1409GVIINVKCKIARQCLEPCKKAGMRFGKCMNGKCHCTPK 1410GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK 1411GVIINVKCKISRACLEPCKKAGMRFGKCMNGKCHCTPK 1412GVIINVKCKISRQCAEPCKKAGMRFGKCMNGKCHCTPK 1413GVIINVKCKISRQCLAPCKKAGMRFGKCMNGKCHCTPK 1414GVIINVKCKISRQCLEACKKAGMRFGKCMNGKCHCTPK 1415GVIINVKCKISRQCLEPCAKAGMRFGKCMNGKCHCTPK 1416GVIINVKCKISRQCLEPCKAAGMRFGKCMNGKCHCTPK 1417GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1418GVIINVKCKISRQCLEPCKKAAMRFGKCMNGKCHCTPK 1419GVIINVKCKISRQCLEPCKKAGARFGKCMNGKCHCTPK 1420GVIINVKCKISRQCLEPCKKAGMAFGKCMNGKCHCTPK 1421GVIINVKCKISRQCLEPCKKAGMRAGKCMNGKCHCTPK 1422GVIINVKCKISRQCLEPCKKAGMRFAKCMNGKCHCTPK 1423GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK 1424GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK 1425GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK 1426GVIINVKCKISRQCLEPCKKAGMRFGKCMNAKCHCTPK 1427GVIINVKCKISRQCLEPCKKAGMRFGKCMNGACHCTPK 1428GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK 1429GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCAPK 1430GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTAK 1431GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPA 1432RVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1433GRIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1434GVRINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1435GVIRNVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1436GVIIRVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1437GVIINRKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1438GVIINVRCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1439GVIINVKCRISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1440GVIINVKCKRSRQCLEPCKKAGMRFGKCMNGKCHCTPK 1441GVIINVKCKIRRQCLEPCKKAGMRFGKCMNGKCHCTPK 1442GVIINVKCKISRRCLEPCKKAGMRFGKCMNGKCHCTPK 1443GVIINVKCKISRQCREPCKKAGMRFGKCMNGKCHCTPK 1444GVIINVKCKISRQCLRPCKKAGMRFGKCMNGKCHCTPK 1445GVIINVKCKISRQCLERCKKAGMRFGKCMNGKCHCTPK 1446GVIINVKCKISRQCLEPCRKAGMRFGKCMNGKCHCTPK 1447GVIINVKCKISRQCLEPCKRAGMRFGKCMNGKCHCTPK 1448GVIINVKCKISRQCLEPCKKRGMRFGKCMNGKCHCTPK 1449GVIINVKCKISRQCLEPCKKARMRFGKCMNGKCHCTPK 1450GVIINVKCKISRQCLEPCKKAGRRFGKCMNGKCHCTPK 1451GVIINVKCKISRQCLEPCKKAGMRRGKCMNGKCHCTPK 1452GVIINVKCKISRQCLEPCKKAGMRFRKCMNGKCHCTPK 1453GVIINVKCKISRQCLEPCKKAGMRFGRCMNGKCHCTPK 1454GVIINVKCKISRQCLEPCKKAGMRFGKCRNGKCHCTPK 1455GVIINVKCKISRQCLEPCKKAGMRFGKCMRGKCHCTPK 1456GVIINVKCKISRQCLEPCKKAGMRFGKCMNRKCHCTPK 1457GVIINVKCKISRQCLEPCKKAGMRFGKCMNGRCHCTPK 1458GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCRCTPK 1459GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCRPK 1460GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTRK 1461GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPR 1462EVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1463GEIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1464GVEINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1465GVIENVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1466GVIIEVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1467GVIINEKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1468GVIINVECKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1469GVIINVKCEISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1470GVIINVKCKESRQCLEPCKKAGMRFGKCMNGKCHCTPK 1471GVIINVKCKIERQCLEPCKKAGMRFGKCMNGKCHCTPK 1472GVIINVKCKISEQCLEPCKKAGMRFGKCMNGKCHCTPK 1473GVIINVKCKISRECLEPCKKAGMRFGKCMNGKCHCTPK 1474GVIINVKCKISRQCEEPCKKAGMRFGKCMNGKCHCTPK 1475GVIINVKCKISRQCLEECKKAGMRFGKCMNGKCHCTPK 1476GVIINVKCKISRQCLEPCEKAGMRFGKCMNGKCHCTPK 1477GVIINVKCKISRQCLEPCKEAGMRFGKCMNGKCHCTPK 1478GVIINVKCKISRQCLEPCKKEGMRFGKCMNGKCHCTPK 1479GVIINVKCKISRQCLEPCKKAEMRFGKCMNGKCHCTPK 1480GVIINVKCKISRQCLEPCKKAGERFGKCMNGKCHCTPK 1481GVIINVKCKISRQCLEPCKKAGMEFGKCMNGKCHCTPK 1482GVIINVKCKISRQCLEPCKKAGMREGKCMNGKCHCTPK 1483GVIINVKCKISRQCLEPCKKAGMRFEKCMNGKCHCTPK 1484GVIINVKCKISRQCLEPCKKAGMRFGECMNGKCHCTPK 1485GVIINVKCKISRQCLEPCKKAGMRFGKCENGKCHCTPK 1486GVIINVKCKISRQCLEPCKKAGMRFGKCMEGKCHCTPK 1487GVIINVKCKISRQCLEPCKKAGMRFGKCMNEKCHCTPK 1488GVIINVKCKISRQCLEPCKKAGMRFGKCMNGECHCTPK 1489GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCECTPK 1490GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCEPK 1491GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTEK 1492GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPE 1493[1-Nal]VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1494G[1-Nal]IINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1495GV[1-Nal]INVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1496GVI[1-Nal]NVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1497GVII[1-Nal]VKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1498GVIIN[1-Nal]KCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1499GVIINV[1-Nal]CKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1500GVIINVKC[1-Nal]ISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1501GVIINVKCK[1-Nal]SRQCLEPCKKAGMRFGKCMNGKCHCTPK 1502GVIINVKCKI[1-Nal]RQCLEPCKKAGMRFGKCMNGKCHCTPK 1503GVIINVKCKIS[1-Nal]QCLEPCKKAGMRFGKCMNGKCHCTPK 1504GVIINVKCKISR[1-Nal]CLEPCKKAGMRFGKCMNGKCHCTPK 1505GVIINVKCKISRQC[1-Nal]EPCKKAGMRFGKCMNGKCHCTPK 1506GVIINVKCKISRQCL[1-Nal]PCKKAGMRFGKCMNGKCHCTPK 1507GVIINVKCKISRQCLE[1-Nal]CKKAGMRFGKCMNGKCHCTPK 1508GVIINVKCKISRQCLEPC[1-Nal]KAGMRFGKCMNGKCHCTPK 1509GVIINVKCKISRQCLEPCK[1-Nal]AGMRFGKCMNGKCHCTPK 1510GVIINVKCKISRQCLEPCKK[1-Nal]GMRFGKCMNGKCHCTPK 1511GVIINVKCKISRQCLEPCKKA[1-Nal]MRFGKCMNGKCHCTPK 1512GVIINVKCKISRQCLEPCKKAG[1-Nal]RFGKCMNGKCHCTPK 1513GVIINVKCKISRQCLEPCKKAGM[1-Nal]FGKCMNGKCHCTPK 1514GVIINVKCKISRQCLEPCKKAGMR[1-Nal]GKCMNGKCHCTPK 1515GVIINVKCKISRQCLEPCKKAGMRF[1-Nal]KCMNGKCHCTPK 1516GVIINVKCKISRQCLEPCKKAGMRFG[1-Nal]CMNGKCHCTPK 1517GVIINVKCKISRQCLEPCKKAGMRFGKC[1-Nal]NGKCHCTPK 1518GVIINVKCKISRQCLEPCKKAGMRFGKCM[1-Nal]GKCHCTPK 1519GVIINVKCKISRQCLEPCKKAGMRFGKCMN[1-Nal]KCHCTPK 1520GVIINVKCKISRQCLEPCKKAGMRFGKCMNG[1-Nal]CHCTPK 1521GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKC[1-Nal]CTPK 1522GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC[1-Nal]PK 1523GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCT[1-Nal]K 1524GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTP[1-Nal] 1525Ac-AVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1526Ac-GAIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1527Ac-GVAINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1528Ac-GVIANVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1529Ac-GVIIAVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1530Ac-GVIINAKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1531Ac-GVIINVACKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1532Ac-GVIINVKCAISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1533Ac-GVIINVKCKASRQCLEPCKKAGMRFGKCMNGKCHCTPK 1534Ac-GVIINVKCKIARQCLEPCKKAGMRFGKCMNGKCHCTPK 1535Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK 1536Ac-GVIINVKCKISRACLEPCKKAGMRFGKCMNGKCHCTPK 1537Ac-GVIINVKCKISRQCAEPCKKAGMRFGKCMNGKCHCTPK 1538Ac-GVIINVKCKISRQCLAPCKKAGMRFGKCMNGKCHCTPK 1539Ac-GVIINVKCKISRQCLEACKKAGMRFGKCMNGKCHCTPK 1540Ac-GVIINVKCKISRQCLEPCAKAGMRFGKCMNGKCHCTPK 1541Ac-GVIINVKCKISRQCLEPCKAAGMRFGKCMNGKCHCTPK 1542Ac-GVIINVKCKISRQCLEPCKKAAMRFGKCMNGKCHCTPK 1543Ac-GVIINVKCKISRQCLEPCKKAGARFGKCMNGKCHCTPK 1544Ac-GVIINVKCKISRQCLEPCKKAGMAFGKCMNGKCHCTPK 1545Ac-GVIINVKCKISRQCLEPCKKAGMRAGKCMNGKCHCTPK 1546Ac-GVIINVKCKISRQCLEPCKKAGMRFAKCMNGKCHCTPK 1547Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK 1548Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK 1549Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK 1550Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNAKCHCTPK 1551Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGACHCTPK 1552Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK 1553Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCAPK 1554Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTAK 1555Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPA 1556Ac-RVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1557Ac-GRIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1558Ac-GVRINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1559Ac-GVIRNVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1560Ac-GVIIRVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1561Ac-GVIINRKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1562Ac-GVIINVRCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1563Ac-GVIINVKCRISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1564Ac-GVIINVKCKRSRQCLEPCKKAGMRFGKCMNGKCHCTPK 1565Ac-GVIINVKCKIRRQCLEPCKKAGMRFGKCMNGKCHCTPK 1566Ac-GVIINVKCKISRRCLEPCKKAGMRFGKCMNGKCHCTPK 1567Ac-GVIINVKCKISRQCREPCKKAGMRFGKCMNGKCHCTPK 1568Ac-GVIINVKCKISRQCLRPCKKAGMRFGKCMNGKCHCTPK 1569Ac-GVIINVKCKISRQCLERCKKAGMRFGKCMNGKCHCTPK 1570Ac-GVIINVKCKISRQCLEPCRKAGMRFGKCMNGKCHCTPK 1571Ac-GVIINVKCKISRQCLEPCKRAGMRFGKCMNGKCHCTPK 1572Ac-GVIINVKCKISRQCLEPCKKRGMRFGKCMNGKCHCTPK 1573Ac-GVIINVKCKISRQCLEPCKKARMRFGKCMNGKCHCTPK 1574Ac-GVIINVKCKISRQCLEPCKKAGRRFGKCMNGKCHCTPK 1575Ac-GVIINVKCKISRQCLEPCKKAGMRRGKCMNGKCHCTPK 1576Ac-GVIINVKCKISRQCLEPCKKAGMRFRKCMNGKCHCTPK 1577Ac-GVIINVKCKISRQCLEPCKKAGMRFGRCMNGKCHCTPK 1578Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCRNGKCHCTPK 1579Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMRGKCHCTPK 1580Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNRKCHCTPK 1581Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGRCHCTPK 1582Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCRCTPK 1583Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCRPK 1584Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTRK 1585Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPR 1586Ac-EVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1587Ac-GEIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1588Ac-GVEINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1589Ac-GVIENVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1590Ac-GVIIEVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1591Ac-GVIINEKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1592Ac-GVIINVECKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1593Ac-GVIINVKCEISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1594Ac-GVIINVKCKESRQCLEPCKKAGMRFGKCMNGKCHCTPK 1595Ac-GVIINVKCKIERQCLEPCKKAGMRFGKCMNGKCHCTPK 1596Ac-GVIINVKCKISEQCLEPCKKAGMRFGKCMNGKCHCTPK 1597Ac-GVIINVKCKISRECLEPCKKAGMRFGKCMNGKCHCTPK 1598Ac-GVIINVKCKISRQCEEPCKKAGMRFGKCMNGKCHCTPK 1599Ac-GVIINVKCKISRQCLEECKKAGMRFGKCMNGKCHCTPK 1600Ac-GVIINVKCKISRQCLEPCEKAGMRFGKCMNGKCHCTPK 1601Ac-GVIINVKCKISRQCLEPCKEAGMRFGKCMNGKCHCTPK 1602Ac-GVIINVKCKISRQCLEPCKKEGMRFGKCMNGKCHCTPK 1603Ac-GVIINVKCKISRQCLEPCKKAEMRFGKCMNGKCHCTPK 1604Ac-GVIINVKCKISRQCLEPCKKAGERFGKCMNGKCHCTPK 1605Ac-GVIINVKCKISRQCLEPCKKAGMEFGKCMNGKCHCTPK 1606Ac-GVIINVKCKISRQCLEPCKKAGMREGKCMNGKCHCTPK 1607Ac-GVIINVKCKISRQCLEPCKKAGMRFEKCMNGKCHCTPK 1608Ac-GVIINVKCKISRQCLEPCKKAGMRFGECMNGKCHCTPK 1609Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCENGKCHCTPK 1610Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMEGKCHCTPK 1611Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNEKCHCTPK 1612Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGECHCTPK 1613Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCECTPK 1614Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCEPK 1615Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTEK 1616Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPE 1617Ac-[1-Nal]VIINVKCKISRQCLEPCKKAGMRFGKCMNGK CHCTPK 1618Ac-G[1-Nal]IINVKCKISRQCLEPCKKAGMRFGKCMNGK CHCTPK 1619Ac-GV[1-Nal]INVKCKISRQCLEPCKKAGMRFGKCMNGK CHCTPK 1620Ac-GVI[1-Nal]NVKCKISRQCLEPCKKAGMRFGKCMNGK CHCTPK 1621Ac-GVII[1-Nal]VKCKISRQCLEPCKKAGMRFGKCMNGK CHCTPK 1622Ac-GVIIN[1-Nal]KCKISRQCLEPCKKAGMRFGKCMNGK CHCTPK 1623Ac-GVIINV[1-Nal]CKISRQCLEPCKKAGMRFGKCMNGK CHCTPK 1624Ac-GVIINVKC[1-Nal]ISRQCLEPCKKAGMRFGKCMNGK CHCTPK 1625Ac-GVIINVKCK[1-Nal]SRQCLEPCKKAGMRFGKCMNGK CHCTPK 1626Ac-GVIINVKCKI[1-Nal]RQCLEPCKKAGMRFGKCMNGK CHCTPK 1627Ac-GVIINVKCKIS[1-Nal]QCLEPCKKAGMRFGKCMNGK CHCTPK 1628Ac-GVIINVKCKISR[1-Nal]CLEPCKKAGMRFGKCMNGK CHCTPK 1629Ac-GVIINVKCKISRQC[1-Nal]EPCKKAGMRFGKCMNGK CHCTPK 1630Ac-GVIINVKCKISRQCL[1-Nal]PCKKAGMRFGKCMNGK CHCTPK 1631Ac-GVIINVKCKISRQCLE[1-Nal]CKKAGMRFGKCMNGK CHCTPK 1632Ac-GVIINVKCKISRQCLEPC[1-Nal]KAGMRFGKCMNGK CHCTPK 1633Ac-GVIINVKCKISRQCLEPCK[1-Nal]AGMRFGKCMNGK CHCTPK 1634Ac-GVIINVKCKISRQCLEPCKK[1-Nal]GMRFGKCMNGK CHCTPK 1635Ac-GVIINVKCKISRQCLEPCKKA[1-Nal]MRFGKCMNGK CHCTPK 1636Ac-GVIINVKCKISRQCLEPCKKAG[1-Nal]RFGKCMNGK CHCTPK 1637Ac-GVIINVKCKISRQCLEPCKKAGM[1-Nal]FGKCMNGK CHCTPK 1638Ac-GVIINVKCKISRQCLEPCKKAGMR[1-Nal]GKCMNGK CHCTPK 1639Ac-GVIINVKCKISRQCLEPCKKAGMRF[1-Nal]KCMNGK CHCTPK 1640Ac-GVIINVKCKISRQCLEPCKKAGMRFG[1-Nal]CMNGK CHCTPK 1641Ac-GVIINVKCKISRQCLEPCKKAGMRFGKC[1-Nal]NGK CHCTPK 1642Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCM[1-Nal]GK CHCTPK 1643Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMN[1-Nal]K CHCTPK 1644Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNG[1-Nal] CHCTPK 1645Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKC [1-Nal]CTPK 1646Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC [1-Nal]PK 1647Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCT [1-Nal]K 1648Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTP [1-Nal] 1649AVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1650GAIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1651GVAINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1652GVIANVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1653GVIIAVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1654GVIINAKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1655GVIINVACKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1656GVIINVKCAISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1657GVIINVKCKASRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1658GVIINVKCKIARQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1659GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1660GVIINVKCKISRACLEPCKKAGMRFGKCMNGKCHCTPK-amide 1661GVIINVKCKISRQCAEPCKKAGMRFGKCMNGKCHCTPK-amide 1662GVIINVKCKISRQCLAPCKKAGMRFGKCMNGKCHCTPK-amide 1663GVIINVKCKISRQCLEACKKAGMRFGKCMNGKCHCTPK-amide 1664GVIINVKCKISRQCLEPCAKAGMRFGKCMNGKCHCTPK-amide 1665GVIINVKCKISRQCLEPCKAAGMRFGKCMNGKCHCTPK-amide 1666GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1667GVIINVKCKISRQCLEPCKKAAMRFGKCMNGKCHCTPK-amide 1668GVIINVKCKISRQCLEPCKKAGARFGKCMNGKCHCTPK-amide 1669GVIINVKCKISRQCLEPCKKAGMAFGKCMNGKCHCTPK-amide 1670GVIINVKCKISRQCLEPCKKAGMRAGKCMNGKCHCTPK-amide 1671GVIINVKCKISRQCLEPCKKAGMRFAKCMNGKCHCTPK-amide 1672GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK-amide 1673GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK-amide 1674GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK-amide 1675GVIINVKCKISRQCLEPCKKAGMRFGKCMNAKCHCTPK-amide 1676GVIINVKCKISRQCLEPCKKAGMRFGKCMNGACHCTPK-amide 1677GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-amide 1678GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCAPK-amide 1679GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTAK-amide 1680GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPA-amide 1681RVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1682GRIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1683GVRINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1684GVIRNVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1685GVIIRVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1686GVIINRKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1687GVIINVRCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1688GVIINVKCRISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1689GVIINVKCKRSRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1690GVIINVKCKIRRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1691GVIINVKCKISRRCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1692GVIINVKCKISRQCREPCKKAGMRFGKCMNGKCHCTPK-amide 1693GVIINVKCKISRQCLRPCKKAGMRFGKCMNGKCHCTPK-amide 1694GVIINVKCKISRQCLERCKKAGMRFGKCMNGKCHCTPK-amide 1695GVIINVKCKISRQCLEPCRKAGMRFGKCMNGKCHCTPK-amide 1696GVIINVKCKISRQCLEPCKRAGMRFGKCMNGKCHCTPK-amide 1697GVIINVKCKISRQCLEPCKKRGMRFGKCMNGKCHCTPK-amide 1698GVIINVKCKISRQCLEPCKKARMRFGKCMNGKCHCTPK-amide 1699GVIINVKCKISRQCLEPCKKAGRRFGKCMNGKCHCTPK-amide 1700GVIINVKCKISRQCLEPCKKAGMRRGKCMNGKCHCTPK-amide 1701GVIINVKCKISRQCLEPCKKAGMRFRKCMNGKCHCTPK-amide 1702GVIINVKCKISRQCLEPCKKAGMRFGRCMNGKCHCTPK-amide 1703GVIINVKCKISRQCLEPCKKAGMRFGKCRNGKCHCTPK-amide 1704GVIINVKCKISRQCLEPCKKAGMRFGKCMRGKCHCTPK-amide 1705GVIINVKCKISRQCLEPCKKAGMRFGKCMNRKCHCTPK-amide 1706GVIINVKCKISRQCLEPCKKAGMRFGKCMNGRCHCTPK-amide 1707GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCRCTPK-amide 1708GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCRPK-amide 1709GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTRK-amide 1710GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPR-amide 1711EVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1712GEIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1713GVEINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1714GVIENVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1715GVIIEVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1716GVIINEKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1717GVIINVECKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1718GVIINVKCEISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1719GVIINVKCKESRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1720GVIINVKCKIERQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1721GVIINVKCKISEQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1722GVIINVKCKISRECLEPCKKAGMRFGKCMNGKCHCTPK-amide 1723GVIINVKCKISRQCEEPCKKAGMRFGKCMNGKCHCTPK-amide 1724GVIINVKCKISRQCLEECKKAGMRFGKCMNGKCHCTPK-amide 1725GVIINVKCKISRQCLEPCEKAGMRFGKCMNGKCHCTPK-amide 1726GVIINVKCKISRQCLEPCKEAGMRFGKCMNGKCHCTPK-amide 1727GVIINVKCKISRQCLEPCKKEGMRFGKCMNGKCHCTPK-amide 1728GVIINVKCKISRQCLEPCKKAEMRFGKCMNGKCHCTPK-amide 1729GVIINVKCKISRQCLEPCKKAGERFGKCMNGKCHCTPK-amide 1730GVIINVKCKISRQCLEPCKKAGMEFGKCMNGKCHCTPK-amide 1731GVIINVKCKISRQCLEPCKKAGMREGKCMNGKCHCTPK-amide 1732GVIINVKCKISRQCLEPCKKAGMRFEKCMNGKCHCTPK-amide 1733GVIINVKCKISRQCLEPCKKAGMRFGECMNGKCHCTPK-amide 1734GVIINVKCKISRQCLEPCKKAGMRFGKCENGKCHCTPK-amide 1735GVIINVKCKISRQCLEPCKKAGMRFGKCMEGKCHCTPK-amide 1736GVIINVKCKISRQCLEPCKKAGMRFGKCMNEKCHCTPK-amide 1737GVIINVKCKISRQCLEPCKKAGMRFGKCMNGECHCTPK-amide 1738GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCECTPK-amide 1739GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCEPK-amide 1740GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTEK-amide 1741GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPE-amide 1742[1-Nal]VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1743G[1-Nal]IINVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1744GV[1-Nal]INVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1745GVI[1-Nal]NVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1746GVII[1-Nal]VKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1747GVIIN[1-Nal]KCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1748GVIINV[1-Nal]CKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1749GVIINVKC[1-Nal]ISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1750GVIINVKCK[1-Nal]SRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1751GVIINVKCKI[1-Nal]RQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1752GVIINVKCKIS[1-Nal]QCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1753GVIINVKCKISR[1-Nal]CLEPCKKAGMRFGKCMNGKCH CTPK-amide 1754GVIINVKCKISRQC[1-Nal]EPCKKAGMRFGKCMNGKCH CTPK-amide 1755GVIINVKCKISRQCL[1-Nal]PCKKAGMRFGKCMNGKCH CTPK-amide 1756GVIINVKCKISRQCLE[1-Nal]CKKAGMRFGKCMNGKCH CTPK-amide 1757GVIINVKCKISRQCLEPC[1-Nal]KAGMRFGKCMNGKCH CTPK-amide 1758GVIINVKCKISRQCLEPCK[1-Nal]AGMRFGKCMNGKCH CTPK-amide 1759GVIINVKCKISRQCLEPCKK[1-Nal]GMRFGKCMNGKCH CTPK-amide 1760GVIINVKCKISRQCLEPCKKA[1-Nal]MRFGKCMNGKCH CTPK-amide 1761GVIINVKCKISRQCLEPCKKAG[1-Nal]RFGKCMNGKCH CTPK-amide 1762GVIINVKCKISRQCLEPCKKAGM[1-Nal]FGKCMNGKCH CTPK-amide 1763GVIINVKCKISRQCLEPCKKAGMR[1-Nal]GKCMNGKCH CTPK-amide 1764GVIINVKCKISRQCLEPCKKAGMRF[1-Nal]KCMNGKCH CTPK-amide 1765GVIINVKCKISRQCLEPCKKAGMRFG[1-Nal]CMNGKCH CTPK-amide 1766GVIINVKCKISRQCLEPCKKAGMRFGKC[1-Nal]NGKCH CTPK-amide 1767GVIINVKCKISRQCLEPCKKAGMRFGKCM[1-Nal]GKCH CTPK-amide 1768GVIINVKCKISRQCLEPCKKAGMRFGKCMN[1-Nal]KCH CTPK-amide 1769GVIINVKCKISRQCLEPCKKAGMRFGKCMNG[1-Nal]CH CTPK-amide 1770GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKC[1-Nal] CTPK-amide 1771GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC [1-Nal]PK-amide 1772GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCT [1-Nal]K-amide 1773GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC TP[1-Nal]-amide 1774Ac-AVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1775Ac-GAIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1776Ac-GVAINVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1777Ac-GVIANVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1778Ac-GVIIAVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1779Ac-GVIINAKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1780Ac-GVIINVACKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1781Ac-GVIINVKCAISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1782Ac-GVIINVKCKASRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1783Ac-GVIINVKCKIARQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1784Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1785Ac-GVIINVKCKISRACLEPCKKAGMRFGKCMNGKCH CTPK-amide 1786Ac-GVIINVKCKISRQCAEPCKKAGMRFGKCMNGKCH CTPK-amide 1787Ac-GVIINVKCKISRQCLAPCKKAGMRFGKCMNGKCH CTPK-amide 1788Ac-GVIINVKCKISRQCLEACKKAGMRFGKCMNGKCH CTPK-amide 1789Ac-GVIINVKCKISRQCLEPCAKAGMRFGKCMNGKCH CTPK-amide 1790Ac-GVIINVKCKISRQCLEPCKAAGMRFGKCMNGKCH CTPK-amide 1791Ac-GVIINVKCKISRQCLEPCKKAAMRFGKCMNGKCH CTPK-amide 1792Ac-GVIINVKCKISRQCLEPCKKAGARFGKCMNGKCH CTPK-amide 1793Ac-GVIINVKCKISRQCLEPCKKAGMAFGKCMNGKCH CTPK-amide 1794Ac-GVIINVKCKISRQCLEPCKKAGMRAGKCMNGKCH CTPK-amide 1795Ac-GVIINVKCKISRQCLEPCKKAGMRFAKCMNGKCH CTPK-amide 1796Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCH CTPK-amide 1797Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCH CTPK-amide 1798Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCH CTPK-amide 1799Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNAKCH CTPK-amide 1800Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGACH CTPK-amide 1801Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCA CTPK-amide 1802Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH CAPK-amide 1803Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTAK-amide 1804Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPA-amide 1805Ac-RVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1806Ac-GRIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1807Ac-GVRINVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1808Ac-GVIRNVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1809Ac-GVIIRVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1810Ac-GVIINRKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1811Ac-GVIINVRCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1812Ac-GVIINVKCRISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1813Ac-GVIINVKCKRSRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1814Ac-GVIINVKCKIRRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1815Ac-GVIINVKCKISRRCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1816Ac-GVIINVKCKISRQCREPCKKAGMRFGKCMNGKCH CTPK-amide 1817Ac-GVIINVKCKISRQCLRPCKKAGMRFGKCMNGKCH CTPK-amide 1818Ac-GVIINVKCKISRQCLERCKKAGMRFGKCMNGKCH CTPK-amide 1819Ac-GVIINVKCKISRQCLEPCRKAGMRFGKCMNGKCH CTPK-amide 1820Ac-GVIINVKCKISRQCLEPCKRAGMRFGKCMNGKCH CTPK-amide 1821Ac-GVIINVKCKISRQCLEPCKKRGMRFGKCMNGKCH CTPK-amide 1822Ac-GVIINVKCKISRQCLEPCKKARMRFGKCMNGKCH CTPK-amide 1823Ac-GVIINVKCKISRQCLEPCKKAGRRFGKCMNGKCH CTPK-amide 1824Ac-GVIINVKCKISRQCLEPCKKAGMRRGKCMNGKCH CTPK-amide 1825Ac-GVIINVKCKISRQCLEPCKKAGMRFRKCMNGKCH CTPK-amide 1826Ac-GVIINVKCKISRQCLEPCKKAGMRFGRCMNGKCH CTPK-amide 1827Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCRNGKCH CTPK-amide 1828Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMRGKCH CTPK-amide 1829Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNRKCH CTPK-amide 1830Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGRCH CTPK-amide 1831Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCR CTPK-amide 1832Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH CRPK-amide 1833Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTRK-amide 1834Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPR-amide 1835Ac-EVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1836Ac-GEIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1837Ac-GVEINVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1838Ac-GVIENVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1839Ac-GVIIEVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1840Ac-GVIINEKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1841Ac-GVIINVECKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1842Ac-GVIINVKCEISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1843Ac-GVIINVKCKESRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1844Ac-GVIINVKCKIERQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1845Ac-GVIINVKCKISEQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1846Ac-GVIINVKCKISRECLEPCKKAGMRFGKCMNGKCH CTPK-amide 1847Ac-GVIINVKCKISRQCEEPCKKAGMRFGKCMNGKCH CTPK-amide 1848Ac-GVIINVKCKISRQCLEECKKAGMRFGKCMNGKCH CTPK-amide 1849Ac-GVIINVKCKISRQCLEPCEKAGMRFGKCMNGKCH CTPK-amide 1850Ac-GVIINVKCKISRQCLEPCKEAGMRFGKCMNGKCH CTPK-amide 1851Ac-GVIINVKCKISRQCLEPCKKEGMRFGKCMNGKCH CTPK-amide 1852Ac-GVIINVKCKISRQCLEPCKKAEMRFGKCMNGKCH CTPK-amide 1853Ac-GVIINVKCKISRQCLEPCKKAGERFGKCMNGKCH CTPK-amide 1854Ac-GVIINVKCKISRQCLEPCKKAGMEFGKCMNGKCH CTPK-amide 1855Ac-GVIINVKCKISRQCLEPCKKAGMREGKCMNGKCH CTPK-amide 1856Ac-GVIINVKCKISRQCLEPCKKAGMRFEKCMNGKCH CTPK-amide 1857Ac-GVIINVKCKISRQCLEPCKKAGMRFGECMNGKCH CTPK-amide 1858Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCENGKCH CTPK-amide 1859Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMEGKCH CTPK-amide 1860Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNEKCH CTPK-amide 1861Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGECH CTPK-amide 1862Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCE CTPK-amide 1863Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH CEPK-amide 1864Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTEK-amide 1865Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPE-amide 1866Ac-[1-Nal]VIINVKCKISRQCLEPCKKAGMRFGKCMNGK CHCTPK-amide 1867Ac-G[1-Nal]IINVKCKISRQCLEPCKKAGMRFGKCMNGK CHCTPK-amide 1868Ac-GV[1-Nal]INVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1869Ac-GVI[1-Nal]NVKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1870Ac-GVII[1-Nal]VKCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1871Ac-GVIIN[1-Nal]KCKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1872Ac-GVIINV[1-Nal]CKISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1873Ac-GVIINVKC[1-Nal]ISRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1874Ac-GVIINVKCK[1-Nal]SRQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1875Ac-GVIINVKCKI[1-Nal]RQCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1876Ac-GVIINVKCKIS[1-Nal]QCLEPCKKAGMRFGKCMNGKCH CTPK-amide 1877Ac-GVIINVKCKISR[1-Nal]CLEPCKKAGMRFGKCMNGKCH CTPK-amide 1878Ac-GVIINVKCKISRQC[1-Nal]EPCKKAGMRFGKCMNGKCH CTPK-amide 1879Ac-GVIINVKCKISRQCL[1-Nal]PCKKAGMRFGKCMNGKCH CTPK-amide 1880Ac-GVIINVKCKISRQCLE[1-Nal]CKKAGMRFGKCMNGKCH CTPK-amide 1881Ac-GVIINVKCKISRQCLEPC[1-Nal]KAGMRFGKCMNGKCH CTPK-amide 1882Ac-GVIINVKCKISRQCLEPCK[1-Nal]AGMRFGKCMNGKCH CTPK-amide 1883Ac-GVIINVKCKISRQCLEPCKK[1-Nal]GMRFGKCMNGKCH CTPK-amide 1884Ac-GVIINVKCKISRQCLEPCKKA[1-Nal]MRFGKCMNGKCH CTPK-amide 1885Ac-GVIINVKCKISRQCLEPCKKAG[1-Nal]RFGKCMNGKCH CTPK-amide 1886Ac-GVIINVKCKISRQCLEPCKKAGM[1-Nal]FGKCMNGKCH CTPK-amide 1887Ac-GVIINVKCKISRQCLEPCKKAGMR[1-Nal]GKCMNGKCH CTPK-amide 1888Ac-GVIINVKCKISRQCLEPCKKAGMRF[1-Nal]KCMNGKCH CTPK-amide 1889Ac-GVIINVKCKISRQCLEPCKKAGMRFG[1-Nal]CMNGKCH CTPK-amide 1890Ac-GVIINVKCKISRQCLEPCKKAGMRFGKC[1-Nal]NGKCH CTPK-amide 1891Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCM[1-Nal]GKCH CTPK-amide 1892Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMN[1-Nal]KCH CTPK-amide 1893Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNG[1-Nal]CH CTPK-amide 1894Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKC[1-Nal] CTPK-amide 1895Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC [1-Nal]PK-amide 1896Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCT [1-Nal]K-amide 1897Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTP [1-Nal]-amide

TABLE 7B Additional useful OSK1 peptide analogsequences: Ala-12 Substituted Series SEQ ID Sequence/structure NO:GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK 1898GVIINVSCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK 1899GVIINVKCKISAQCLKPCKKAGMRFGKCMNGKCHCTPK 1900GVIINVKCKISAQCLEPCKDAGMRFGKCMNGKCHCTPK 1901GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1902GVIINVSCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1903GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK 1904GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYPK 1905Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK 1906GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK- 1907 amideAc-GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK- 1908 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYPK-amide 1909Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYPK 1910Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYPK- 1911 amideGVIINVKCKISAQCLKPCKKAGMRFGKCMNGKCHCTPK-amide 1912Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCMNGKCHCTPK 1913Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCMNGKCHCTPK- 1914 amideAc-GVIINVKCKISAQCLEPCKDAGMRFGKCMNGKCHCTPK 1915GVIINVKCKISAQCLEPCKDAGMRFGKCMNGKCHCTPK-amide 1916Ac-GVIINVKCKISAQCLEPCKDAGMRFGKCMNGKCHCTPK- 1917 amideGVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1918Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK 1919Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK- 1920 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide 1921Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1922Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK- 1923 amideVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK 1924Ac-VIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK 1925VIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1926Ac-VIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK- 1927 amideGVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK 1928Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK 1929GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK-amide 1930Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK- 1931 amideVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1932Ac-VIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1933VIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide 1934Ac-VIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK- 1935 amideNVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1936Ac-NVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1937NVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide 1938Ac-NVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide 1939KCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1940Ac-KCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1941KCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide 1942Ac-KCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide 1943CKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1944 Ac-CKISAQCLKPCKDAGMRFGKCMNGKCHCTPK1945 CKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide 1946Ac-CKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide 1947GVIINVKCKISAQCLKPCKDAGMRNGKCMNGKCHCTPK 1948GVIINVKCKISAQCLKPCKDAGMRNGKCMNGKCHCTPK-amide 1949Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCMNGKCHCTPK 1950Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCMNGKCHCTPK- 1951 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK 1952GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-amide 1953Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK 1954Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK- 1955 amideGVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK 1956Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK 1957GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK-amide 1958Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK- 1959 amideTIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1960Ac-TIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1961TIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide 1962Ac-TIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK- 1963 amideGVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK 1964Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK 1965GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1966Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK- 1967 amideGVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK 1968GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1969GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCACTPK 1970Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK 1971GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK-amide 1972Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK- 1973 amideAc-GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCACTPK 1974GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCACTPK-amide 1975Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCACTPK- 1976 amideAc-GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1977GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide 1978Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK- 1979 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCT 1980GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCTPK 1981GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCTPK 1982GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCTPK 1983GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCTPK 1984GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK 1985GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCTPK 1986GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCHCTPK 1987GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCYPK 1988GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCYPK 1989GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCYPK 1990GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCYPK 1991GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCYPK 1992GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCYPK 1993GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCHCYPK 1994GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYPK 1995GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCGCYPK 1996GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACFPK 1997GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACWPK 1998GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCACYPK 1999GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACTPK 2000GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACTPK 2001GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACTPK 2002GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACTPK 2003GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK 2004GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACTPK 2005GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACTPK 2006GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHC 2007GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHC 2008GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHC 2009GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHC 2010GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHC 2011GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHC 2012GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCHC 2013GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCAC 2014GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCAC 2015GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCAC 2016GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCAC 2017GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHC 2018GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCAC 2019GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCAC 2020GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCGCYGG 2021GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCYGG 2022GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCYGG 2023GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCYGG 2024GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCYGG 2025GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCYGG 2026GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCYGG 2027GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYGG 2028GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACYGG 2029GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACYGG 2030GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACYGG 2031GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACYGG 2032GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCYGG 2033GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACYGG 2034GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACYGG 2035GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYG 2036GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCGGG 2037GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCGGG 2038GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCGGG 2039GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCGGG 2040GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCGGG 2041GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCGGG 2042GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACFGG 2043GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACGGG 2044GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACGGG 2045GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACGGG 2046GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACGGG 2047GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCACGGG 2048GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACGGG 2049GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACGGG 2050GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACGG 2051GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYG 2052GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACGG 2053GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCTPK 2054GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCTPK 2055GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCTPK 2056GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK 2057GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCTPK 2058GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCTPK 2059GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCYPK 2060GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCYPK 2061GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCYPK 2062GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCYPK 2063GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYPK 2064GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCYPK 2065GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCYPK 2066GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACTPK 2067GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACTPK 2068GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACTPK 2069GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACTPK 2070GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK 2071GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACTPK 2072GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACTPK 2073GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHC 2074GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHC 2075GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHC 2076GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHC 2077GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHC 2078GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHC 2079GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCAC 2080GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCAC 2081GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCAC 2082GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCAC 2083GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHC 2084GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCAC 2085GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCAC 2086GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCHCYGG 2087GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCYGG 2088GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCHCYG 2089GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCACYG 2090GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCYGG 2091GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCYGG 2092GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCYGG 2093GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG 2094GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCYGG 2095GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCYGG 2096GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCACYG 2097GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACYGG 2098GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACYGG 2099GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACYGG 2100GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACYGG 2101GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG 2102GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACYGG 2103GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACYGG 2104GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCACFGG 2105GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCGGG 2106GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCGGG 2107GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCGGG 2108GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCGGG 2109GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCGGG 2110GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCGGG 2111GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACGGG 2112GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACGGG 2113GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACGGG 2114GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACGGG 2115GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCACTP 2116GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACTP 2117GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACTP 2118GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCTPK-amide 2119GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCTPK- 2120 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCTPK- 2121 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCTPK- 2122 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK- 2123 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCTPK- 2124 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCHCTPK- 2125 amideGVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCYPK-amide 2126GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCYPK- 2127 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCYPK- 2128 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCYPK- 2129 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCYPK- 2130 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCYPK- 2131 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCHCYPK- 2132 amideGVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACTPK-amide 2133GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACTPK- 2134 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACTPK- 2135 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACTPK- 2136 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCACTPK- 2137 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACTPK- 2138 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACTPK- 2139 amideGVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHC-amide 2140GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHC- 2141 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHC- 2142 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHC- 2143 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHC- 2144 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHC- 2145 amideGVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCAC-amide 2146GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCAC- 2147 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCAC- 2148 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCAC- 2149 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHC- 2150 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCAC- 2151 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCAC- 2152 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYGG- 2153 amideGVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCYGG- 2154 amideGVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCYGG- 2155 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCYGG- 2156 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCYGG- 2157 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCYGG- 2158 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCYGG- 2159 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCFGG-amide 2160GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYG-amide 2161GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYG-amide 2162GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACYGG-amide 2163GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACYGG- 2164 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACYGG- 2165 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACYGG- 2166 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCACYGG- 2167 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACYGG- 2168 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACYGG- 2169 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYGG-amide 2170GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCGGG-amide 2171GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCGGG- 2172 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCGGG- 2173 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCGGG- 2174 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCGGG- 2175 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCGGG- 2176 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACGGG-amide 2177GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACFGG-amide 2178GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACGGG-amide 2179GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACGGG- 2180 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACGGG- 2181 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACGGG- 2182 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCACGGG- 2183 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACGGG- 2184 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACGGG- 2185 amideGVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCTPK-amide 2186GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCTPK- 2187 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCTPK- 2188 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCTPK- 2189 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK- 2190 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCTPK- 2191 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCTPK- 2192 amideGVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCYPK-amide 2193GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCYPK- 2194 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCYPK- 2195 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCYPK- 2196 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYPK- 2197 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCYPK- 2198 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCYPK- 2199 amideGVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACTPK-amide 2200GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACTPK- 2201 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACTPK- 2202 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACTPK- 2203 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCACTPK- 2204 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACTPK- 2205 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACTPK- 2206 amideGVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHC-amide 2207GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHC- 2208 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHC- 2209 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHC- 2210 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHC- 2211 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHC- 2212 amideGVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCAC-amide 2213GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCAC- 2214 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCAC- 2215 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCAC- 2216 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHC- 2217 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCAC- 2218 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCAC- 2219 amideGVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCHCWGG- 2220 amideGVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCYGG- 2221 amideGVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCYGG- 2222 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCYGG- 2223 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCYGG- 2224 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG- 2225 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCYGG- 2226 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCYGG- 2227 amideGVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCACYGG-amide 2228GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACYGG-amide 2229GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACYGG- 2230 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACYGG- 2231 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACYGG- 2232 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG- 2233 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACYGG- 2234 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACYGG- 2235 amideGVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCGGG-amide 2236GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCGGG- 2237 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCGGG- 2238 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCGGG- 2239 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCGGG- 2240 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCGGG- 2241 amideGVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACGGG-amide 2242GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACGGG- 2243 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACGGG- 2244 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACGGG- 2245 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCACTP- 2246 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACGGG- 2247 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACGGG- 2248 amideGVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMNGKCACYGG- 2249 amideGVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMNGKCACGGG- 2250 amideGVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMNGKCACY- 2251 amideAc-GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYGG- 2252 amideGVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMNGKCACYGG- 2253 amideGVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMNGKCHCYGG- 2254 amideGVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMNGKCACYGG 2255GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCACYGG-amide 2256GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCACYGG 2257GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCACY-amide 2258GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCHCYGG-amide 2259GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCHCYGG 2260GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCHCYPK 2261GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCAC 2262GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[1Nal]GG- 2263 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[1Nal]PK- 2264 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[2Nal]GG- 2265 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[Cha]GG- 2266 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[MePhe] 2267 GG-amideGVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[BiPhA] 2268 GG-amideGVIINVKCKISAQCLKPCKDAGMRFGKCMNGKC[Aib]CYGG- 2269 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMNGKC[Abu]CYGG- 2270 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[1Nal] 2271GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCAC[1Nal]GG- 2272 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[4Bip]- 2273 amideGVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCAC[4Bip]GG- 2274 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCGGG 2275

TABLE 7C Additional useful OSK1 peptide analog sequences: Ala-12 & Ala27Substituted Series SEQ ID Sequence/structure NO:GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK 2276GVIINVSCKISAQCLEPCKKAGMRFGACMNGKCHCTPK 2277GVIINVKCKISAQCLKPCKKAGMRFGACMNGKCHCTPK 2278GVIINVKCKISAQCLEPCKDAGMRFGACMNGKCHCTPK 2279GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2280GVIINVSCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2281GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK 2282GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYPK 2283Ac-GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK 2284GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK-amide 2285Ac-GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK- 2286 amideGVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYPK-amide 2287Ac-GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYPK 2288Ac-GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYPK- 2289 amideGVIINVKCKISAQCLKPCKKAGMRFGACMNGKCHCTPK-amide 2290Ac-GVIINVKCKISAQCLKPCKKAGMRFGACMNGKCHCTPK 2291Ac-GVIINVKCKISAQCLKPCKKAGMRFGACMNGKCHCTPK- 2292 amideAc-GVIINVKCKISAQCLEPCKDAGMRFGACMNGKCHCTPK 2293GVIINVKCKISAQCLEPCKDAGMRFGACMNGKCHCTPK-amide 2294Ac-GVIINVKCKISAQCLEPCKDAGMRFGACMNGKCHCTPK- 2295 amideGVIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK-amide 2296Ac-GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK 2297Ac-GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK- 2298 amideGVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide 2299Ac-GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2300Ac-GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK- 2301 amideVIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK 2302Ac-VIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK 2303VIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK-amide 2304Ac-VIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK- 2305 amideGVIINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK 2306Ac-GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK 2307GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK-amide 2308Ac-GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK- 2309 amideVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2310Ac-VIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2311VIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide 2312Ac-VIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK- 2313 amideNVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2314Ac-NVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2315NVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide 2316Ac-NVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide 2317KCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2318Ac-KCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2319KCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide 2320Ac-KCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide 2321CKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2322 Ac-CKISAQCLKPCKDAGMRFGACMNGKCHCTPK2323 CKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide 2324Ac-CKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide 2325GVIINVKCKISAQCLKPCKDAGMRNGACMNGKCHCTPK 2326GVIINVKCKISAQCLKPCKDAGMRNGACMNGKCHCTPK-amide 2327Ac-GVIINVKCKISAQCLKPCKDAGMRNGACMNGKCHCTPK 2328Ac-GVIINVKCKISAQCLKPCKDAGMRNGACMNGKCHCTPK- 2329 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK 2330GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-amide 2331Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK 2332Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK- 2333 amideGVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK 2334Ac-GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK 2335GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK-amide 2336Ac-GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK- 2337 amideTIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2338Ac-TIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2339TIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide 2340Ac-TIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK- 2341 amideGVKINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK 2342Ac-GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK 2343GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK-amide 2344Ac-GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK- 2345 amideGVKINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK 2346GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2347GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCACTPK 2348Ac-GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK 2349GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK-amide 2350Ac-GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK- 2351 amideAc-GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCACTPK 2352GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCACTPK-amide 2353Ac-GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCACTPK- 2354 amideAc-GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2355GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide 2356Ac-GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK- 2357 amideGVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCT 2358GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCTPK 2359GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCTPK 2360GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCTPK 2361GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCTPK 2362GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCTPK 2363GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCTPK 2364GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCHCTPK 2365GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCYPK 2366GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCYPK 2367GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCYPK 2368GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCYPK 2369GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCYPK 2370GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCYPK 2371GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCHCYPK 2372GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYPK 2373GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCGCYPK 2374GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACFPK 2375GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACWPK 2376GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCACYPK 2377GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACTPK 2378GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACTPK 2379GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACTPK 2380GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACTPK 2381GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCTPK 2382GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACTPK 2383GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACTPK 2384GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHC 2385GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHC 2386GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHC 2387GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHC 2388GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHC 2389GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHC 2390GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCHC 2391GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCAC 2392GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCAC 2393GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCAC 2394GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCAC 2395GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHC 2396GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCAC 2397GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCAC 2398GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCGCYGG 2399GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCYGG 2400GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCYGG 2401GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCYGG 2402GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCYGG 2403GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCYGG 2404GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCYGG 2405GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYGG 2406GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACYGG 2407GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACYGG 2408GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACYGG 2409GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACYGG 2410GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCYGG 2411GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACYGG 2412GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACYGG 2413GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYG 2415GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCGGG 2416GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCGGG 2417GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCGGG 2418GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCGGG 2419GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCGGG 2420GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCGGG 2421GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACFGG 2422GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACGGG 2423GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACGGG 2424GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACGGG 2425GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACGGG 2426GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCACGGG 2427GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACGGG 2428GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACGGG 2429GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACGG 2430GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYG 2431GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACGG 2432GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCTPK 2433GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCTPK 2434GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCTPK 2435GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCTPK 2436GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCTPK 2437GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCTPK 2438GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCYPK 2439GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCYPK 2440GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCYPK 2441GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCYPK 2442GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYPK 2443GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCYPK 2444GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCYPK 2445GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACTPK 2446GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACTPK 2447GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACTPK 2448GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACTPK 2449GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCTPK 2450GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACTPK 2451GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACTPK 2452GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHC 2453GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHC 2454GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHC 2455GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHC 2456GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHC 2457GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHC 2458GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCAC 2459GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCAC 2460GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCAC 2461GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCAC 2462GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHC 2463GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCAC 2464GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCAC 2465GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCHCYGG 2466GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCYGG 2467GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCHCYG 2468GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCACYG 2469GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCYGG 2470GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCYGG 2471GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCYGG 2472GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYGG 2473GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCYGG 2474GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCYGG 2475GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCACYG 2476GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACYGG 2477GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACYGG 2478GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACYGG 2479GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACYGG 2480GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYGG 2481GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACYGG 2482GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACYGG 2483GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCACFGG 2484GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCGGG 2485GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCGGG 2486GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCGGG 2487GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCGGG 2488GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCGGG 2489GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCGGG 2490GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACGGG 2491GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACGGG 2492GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACGGG 2493GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACGGG 2494GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCACTP 2495GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACTP 2496GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACTP 2497GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCTPK-amide 2498GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCTPK- 2499 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCTPK- 2500 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCTPK- 2501 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCTPK- 2502 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCTPK- 2503 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCHCTPK- 2504 amideGVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCYPK-amide 2505GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCYPK- 2506 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCYPK- 2507 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCYPK- 2508 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCYPK- 2509 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCYPK- 2510 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCHCYPK- 2511 amideGVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACTPK-amide 2512GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACTPK- 2513 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACTPK- 2514 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACTPK- 2515 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCACTPK- 2516 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACTPK- 2517 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACTPK- 2518 amideGVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHC-amide 2519GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHC- 2520 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHC- 2521 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHC- 2522 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHC- 2523 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHC-amide 2524GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCAC-amide 2525GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCAC- 2526 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCAC- 2527 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCAC-amide 2528GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHC- 2529 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCAC- 2530 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCAC-amide 2531GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYGG-amide 2532GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCYGG-amide 2533GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCYGG- 2534 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCYGG- 2535 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCYGG- 2536 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCYGG- 2537 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCYGG- 2538 amideGVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCFGG-amide 2539GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYG-amide 2540GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYG-amide 2541GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACYGG-amide 2542GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACYGG- 2543 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACYGG- 2544 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACYGG- 2545 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCACYGG- 2546 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACYGG- 2547 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACYGG- 2548 amideGVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYGG-amide 2549GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCGGG-amide 2550GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCGGG- 2551 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCGGG- 2552 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCGGG- 2553 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCGGG- 2554 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCGGG- 2555 amideGVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACGGG-amide 2556GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACFGG-amide 2557GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACGGG-amide 2558GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACGGG- 2559 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACGGG- 2560 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACGGG- 2561 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCACGGG- 2562 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACGGG- 2563 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACGGG- 2564 amideGVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCTPK-amide 2565GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCTPK- 2566 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCTPK- 2567 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCTPK- 2568 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCTPK- 2569 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCTPK- 2570 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCTPK- 2571 amideGVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCYPK-amide 2572GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCYPK- 2573 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCYPK- 2574 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCYPK- 2575 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYPK- 2576 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCYPK- 2577 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCYPK- 2578 amideGVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACTPK-amide 2579GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACTPK- 2580 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACTPK- 2581 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACTPK- 2582 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCACTPK- 2583 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACTPK- 2584 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACTPK- 2585 amideGVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHC-amide 2586GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHC- 2587 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHC- 2588 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHC-amide 2589GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHC- 2590 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHC- 2591 amideGVIINVKCKISAQCLOPCKEAGMRFGACMNGKCAC-amide 2592GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCAC- 2593 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCAC- 2594 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCAC-amide 2595GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHC- 2596 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCAC- 2597 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCAC-amide 2598GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCHCWGG-amide 2599GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCYGG-amide 2600GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCYGG- 2601 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCYGG- 2602 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCYGG- 2603 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYGG- 2604 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCYGG- 2605 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCYGG- 2606 amideGVIINVKCKISAQCLKPCKEAGMRFGACMNGKCACYGG-amide 2607GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACYGG-amide 2608GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACYGG- 2609 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACYGG- 2610 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACYGG- 2611 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYGG- 2612 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACYGG- 2613 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACYGG- 2614 amideGVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCGGG-amide 2615GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCGGG- 2616 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCGGG- 2617 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCGGG- 2618 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCGGG- 2619 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCGGG- 2620 amideGVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACGGG-amide 2621GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACGGG- 2622 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACGGG- 2623 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACGGG- 2624 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCACTP- 2625 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACGGG- 2626 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACGGG- 2627 amideGVIINVKCKISAQCLKPCK[Cpa]AGMRFGACMNGKCACYGG- 2628 amideGVIINVKCKISAQCLKPCK[Cpa]AGMRFGACMNGKCACGGG- 2629 amideGVIINVKCKISAQCLKPCK[Cpa]AGMRFGACMNGKCACY- 2630 amideAc-GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYGG- 2631 amideGVIINVKCKISAQCLKPCK[Aad]AGMRFGACMNGKCACYGG- 2632 amideGVIINVKCKISAQCLKPCK[Aad]AGMRFGACMNGKCHCYGG- 2633 amideGVIINVKCKISAQCLKPCK[Aad]AGMRFGACMNGKCACYGG 2634GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCACYGG-amide 2635GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCACYGG 2636GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCACY-amide 2637GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCHCYGG-amide 2638GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCHCYGG 2639GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCHCYPK 2640GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCAC 2641GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[1Nal]GG- 2642 amideGVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[1Nal]PK- 2643 amideGVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[2Nal]GG- 2644 amideGVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[Cha]GG- 2645 amideGVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[MePhe]GG- 2646 amideGVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[BiPhA]GG- 2647 amideGVIINVKCKISAQCLKPCKDAGMRFGACMNGKC[Aib]CYGG- 2648 amideGVIINVKCKISAQCLKPCKDAGMRFGACMNGKC[Abu]CYGG- 2649 amideGVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[1Nal] 2650GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCAC[1Nal]GG- 2651 amideGVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[4Bip]- 2652 amideGVIINVKCKISAQCLHPCKDAGMRFGACMNGKCAC[4Bip]GG- 2653 amideGVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCGGG 2654

TABLE 7D Additional useful OSK1 peptide analog sequences: Ala-12 & Ala29Substituted Series SEQ ID Sequence/structure NO:GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK 2655GVIINVSCKISAQCLEPCKKAGMRFGKCANGKCHCTPK 2656GVIINVKCKISAQCLKPCKKAGMRFGKCANGKCHCTPK 2657GVIINVKCKISAQCLEPCKDAGMRFGKCANGKCHCTPK 2658GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2659GVIINVSCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2660GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK 2661GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYPK 2662Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK 2663GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK-amide 2664Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK- 2665 amideGVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYPK-amide 2666Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYPK 2667Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYPK- 2668 amideGVIINVKCKISAQCLKPCKKAGMRFGKCANGKCHCTPK-amide 2669Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCANGKCHCTPK 2670Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCANGKCHCTPK- 2671 amideAc-GVIINVKCKISAQCLEPCKDAGMRFGKCANGKCHCTPK 2672GVIINVKCKISAQCLEPCKDAGMRFGKCANGKCHCTPK-amide 2673Ac-GVIINVKCKISAQCLEPCKDAGMRFGKCANGKCHCTPK- 2674 amideGVIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK-amide 2675Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK 2676Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK- 2677 amideGVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide 2678Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2679Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK- 2680 amideVIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK 2681Ac-VIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK 2682VIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK-amide 2683Ac-VIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK- 2684 amideGVIINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK 2685Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK 2686GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK-amide 2687Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK- 2688 amideVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2689Ac-VIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2690VIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide 2691Ac-VIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK- 2692 amideNVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2693Ac-NVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2694NVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide 2695Ac-NVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide 2696KCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2697Ac-KCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2698KCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide 2699Ac-KCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide 2700CKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2701 Ac-CKISAQCLKPCKDAGMRFGKCANGKCHCTPK2702 CKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide 2703Ac-CKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide 2704GVIINVKCKISAQCLKPCKDAGMRNGKCANGKCHCTPK 2705GVIINVKCKISAQCLKPCKDAGMRNGKCANGKCHCTPK-amide 2706Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCANGKCHCTPK 2707Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCANGKCHCTPK- 2708 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK 2709GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-amide 2710Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK 2711Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK- 2712 amideGVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK 2713Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK 2714GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK-amide 2715Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK- 2716 amideTIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2717Ac-TIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2718TIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide 2719Ac-TIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK- 2720 amideGVKINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK 2721Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK 2722GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK-amide 2723Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK- 2724 amideGVKINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK 2725GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2726GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCACTPK 2727Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK 2728GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK-amide 2729Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK- 2730 amideAc-GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCACTPK 2731GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCACTPK-amide 2732Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCACTPK- 2733 amideAc-GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2734GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide 2735Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK- 2736 amideGVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCT 2737GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCTPK 2738GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCTPK 2739GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCTPK 2740GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCTPK 2741GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCTPK 2742GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCTPK 2743GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCHCTPK 2744GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCYPK 2745GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCYPK 2746GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCYPK 2747GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCYPK 2748GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCYPK 2749GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCYPK 2750GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCHCYPK 2751GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYPK 2752GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCGCYPK 2753GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACFPK 2754GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACWPK 2755GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCACYPK 2756GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACTPK 2757GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACTPK 2758GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACTPK 2759GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACTPK 2760GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCTPK 2761GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACTPK 2762GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACTPK 2763GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHC 2764GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHC 2765GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHC 2766GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHC 2767GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHC 2768GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHC 2769GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCHC 2770GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCAC 2771GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCAC 2772GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCAC 2773GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCAC 2774GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHC 2775GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCAC 2776GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCAC 2777GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCGCYGG 2778GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCYGG 2779GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCYGG 2780GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCYGG 2781GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCYGG 2782GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCYGG 2783GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCYGG 2784GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYGG 2785GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACYGG 2786GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACYGG 2787GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACYGG 2788GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACYGG 2789GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCYGG 2790GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACYGG 2791GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACYGG 2792GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYG 2793GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCGGG 2794GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCGGG 2795GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCGGG 2796GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCGGG 2797GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCGGG 2798GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCGGG 2799GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACFGG 2800GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACGGG 2801GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACGGG 2802GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACGGG 2803GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACGGG 2804GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCACGGG 2805GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACGGG 2806GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACGGG 2807GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACGG 2808GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYG 2809GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACGG 2810GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCTPK 2811GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCTPK 2812GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCTPK 2813GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCTPK 2814GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCTPK 2815GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCTPK 2816GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCYPK 2817GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCYPK 2818GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCYPK 2819GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCYPK 2820GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYPK 2821GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCYPK 2822GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCYPK 2823GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACTPK 2824GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACTPK 2825GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACTPK 2826GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACTPK 2827GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCTPK 2828GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACTPK 2829GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACTPK 2830GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHC 2831GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHC 2832GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHC 2833GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHC 2834GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHC 2835GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHC 2836GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCAC 2837GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCAC 2838GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCAC 2839GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCAC 2840GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHC 2841GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCAC 2842GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCAC 2843GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCHCYGG 2844GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCYGG 2845GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCHCYG 2846GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCACYG 2847GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCYGG 2848GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCYGG 2849GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCYGG 2850GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYGG 2851GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCYGG 2852GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCYGG 2853GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCACYG 2854GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACYGG 2855GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACYGG 2856GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACYGG 2857GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACYGG 2858GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYGG 2859GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACYGG 2860GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACYGG 2861GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCACFGG 2862GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCGGG 2863GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCGGG 2864GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCGGG 2865GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCGGG 2866GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCGGG 2867GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCGGG 2868GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACGGG 2869GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACGGG 2870GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACGGG 2871GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACGGG 2872GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCACTP 2873GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACTP 2874GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACTP 2875GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCTPK-amide 2876GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCTPK- 2877 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCTPK- 2878 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCTPK- 2879 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCTPK- 2880 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCTPK- 2881 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCHCTPK- 2882 amideGVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCYPK-amide 2883GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCYPK- 2884 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCYPK- 2885 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCYPK- 2886 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCYPK- 2887 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCYPK- 2888 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCHCYPK- 2889 amideGVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACTPK-amide 2890GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACTPK- 2891 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACTPK- 2892 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACTPK- 2893 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCACTPK- 2894 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACTPK- 2895 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACTPK- 2896 amideGVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHC-amide 2897GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHC- 2898 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHC- 2899 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHC-amide 2900GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHC- 2901 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHC-amide 2902GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCAC-amide 2903GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCAC- 2904 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCAC- 2905 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCAC-amide 2906GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHC- 2907 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCAC- 2908 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCAC-amide 2909GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYGG-amide 2910GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCYGG-amide 2911GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCYGG- 2912 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCYGG- 2913 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCYGG- 2914 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCYGG- 2915 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCYGG- 2916 amideGVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCFGG-amide 2917GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYG-amide 2918GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYG-amide 2919GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACYGG-amide 2920GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACYGG- 2921 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACYGG- 2922 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACYGG- 2923 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCACYGG- 2924 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACYGG- 2925 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACYGG- 2926 amideGVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYGG-amide 2927GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCGGG-amide 2928GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCGGG- 2929 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCGGG- 2930 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCGGG- 2931 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCGGG- 2932 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCGGG- 2933 amideGVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACGGG-amide 2934GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACFGG-amide 2935GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACGGG-amide 2936GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACGGG- 2937 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACGGG- 2938 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACGGG- 2939 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCACGGG- 2940 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACGGG- 2941 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACGGG- 2942 amideGVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCTPK-amide 2943GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCTPK- 2944 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCTPK- 2945 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCTPK- 2946 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCTPK- 2947 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCTPK- 2948 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCTPK- 2949 amideGVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCYPK-amide 2950GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCYPK- 2951 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCYPK- 2952 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCYPK- 2953 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYPK- 2954 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCYPK- 2955 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCYPK- 2956 amideGVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACTPK-amide 2957GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACTPK- 2958 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACTPK- 2959 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACTPK- 2960 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCACTPK- 2961 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACTPK- 2962 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACTPK- 2963 amideGVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHC-amide 2964GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHC- 2965 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHC- 2966 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHC-amide 2967GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHC- 2968 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHC-amide 2969GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCAC-amide 2970GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCAC- 2971 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCAC- 2972 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCAC-amide 2973GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHC- 2974 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCAC-amide 2975GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCAC-amide 2976GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCHCWGG-amide 2977GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCYGG-amide 2978GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCYGG- 2979 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCYGG- 2980 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCYGG- 2981 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYGG- 2982 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCYGG- 2983 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCYGG- 2984 amideGVIINVKCKISAQCLKPCKEAGMRFGKCANGKCACYGG-amide 2985GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACYGG-amide 2986GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACYGG- 2987 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACYGG- 2988 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACYGG- 2989 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYGG- 2990 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACYGG- 2991 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACYGG- 2992 amideGVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCGGG-amide 2993GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCGGG- 2994 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCGGG- 2995 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCGGG- 2996 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCGGG- 2997 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCGGG- 2998 amideGVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACGGG-amide 2999GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACGGG- 3000 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACGGG- 3001 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACGGG- 3002 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCACTP- 3003 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACGGG- 3004 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACGGG- 3005 amideGVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCANGKCACYGG- 3006 amideGVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCANGKCACGGG- 3007 amideGVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCANGKCACY- 3008 amideAc-GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYGG- 3009 amideGVIINVKCKISAQCLKPCK[Aad]AGMRFGKCANGKCACYGG- 3010 amideGVIINVKCKISAQCLKPCK[Aad]AGMRFGKCANGKCHCYGG- 3011 amideGVIINVKCKISAQCLKPCK[Aad]AGMRFGKCANGKCACYGG 3012GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCACYGG-amide 3013GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCACYGG 3014GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCACY-amide 3015GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCHCYGG-amide 3016GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCHCYGG 3017GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCHCYPK 3018GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCAC 3019GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[1Nal]GG- 3020 amideGVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[1Nal]PK- 3021 amideGVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[2Nal]GG- 3022 amideGVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[Cha]GG- 3023 amideGVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[MePhe]GG- 3024 amideGVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[BiPhA]GG- 3025 amideGVIINVKCKISAQCLKPCKDAGMRFGKCANGKC[Aib]CYGG- 3026 amideGVIINVKCKISAQCLKPCKDAGMRFGKCANGKC[Abu]CYGG- 3027 amideGVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[1Nal] 3028GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCAC[1Nal]GG- 3029 amideGVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[4Bip]- 3030 amideGVIINVKCKISAQCLHPCKDAGMRFGKCANGKCAC[4Bip]GG- 3031 amideGVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCGGG 3032

TABLE 7E Additional useful OSK1 peptide analogs: Ala-12 & Ala30Substituted Series SEQ ID Sequence/structure NO:GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK 3033GVIINVSCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK 3034GVIINVKCKISAQCLKPCKKAGMRFGKCMAGKCHCTPK 3035GVIINVKCKISAQCLEPCKDAGMRFGKCMAGKCHCTPK 3036GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3037GVIINVSCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3038GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK 3039GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYPK 3040Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK 3041GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 3042Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK- 3043 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYPK-amide 3044Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYPK 3045Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYPK- 3046 amideGVIINVKCKISAQCLKPCKKAGMRFGKCMAGKCHCTPK-amide 3047Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCMAGKCHCTPK 3048Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCMAGKCHCTPK- 3049 amideAc-GVIINVKCKISAQCLEPCKDAGMRFGKCMAGKCHCTPK 3050GVIINVKCKISAQCLEPCKDAGMRFGKCMAGKCHCTPK-amide 3051Ac-GVIINVKCKISAQCLEPCKDAGMRFGKCMAGKCHCTPK- 3052 amideGVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK-amide 3053Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK 3054Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK- 3055 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 3056Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3057Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK- 3058 amideVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK 3059Ac-VIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK 3060VIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK-amide 3061Ac-VIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK- 3062 amideGVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK 3063Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK 3064GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK-amide 3065Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK- 3066 amideVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3067Ac-VIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3068VIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 3069Ac-VIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK- 3070 amideNVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3071Ac-NVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3072NVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 3073Ac-NVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 3074KCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3075Ac-KCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3076KCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 3077Ac-KCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 3078CKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3079 Ac-CKISAQCLKPCKDAGMRFGKCMAGKCHCTPK3080 CKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 3081Ac-CKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 3082GVIINVKCKISAQCLKPCKDAGMRNGKCMAGKCHCTPK 3083GVIINVKCKISAQCLKPCKDAGMRNGKCMAGKCHCTPK-amide 3084Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCMAGKCHCTPK 3085Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCMAGKCHCTPK- 3086 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK 3087GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-amide 3088Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK 3089Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK- 3090 amideGVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK 3091Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK 3092GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK-amide 3093Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK- 3094 amideTIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3095Ac-TIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3096TIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 3097Ac-TIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK- 3098 amideGVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK 3099Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK 3100GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK-amide 3101Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK- 3102 amideGVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK 3103GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3104GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCACTPK 3105Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK 3106GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK-amide 3107Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK- 3108 amideAc-GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCACTPK 3109GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCACTPK-amide 3110Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCACTPK- 3111 amideAc-GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3112GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 3113Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK- 3114 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCT 3115GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCTPK 3116GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCTPK 3117GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCTPK 3118GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCTPK 3119GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK 3120GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCTPK 3121GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCHCTPK 3122GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCYPK 3123GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCYPK 3124GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCYPK 3125GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCYPK 3126GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCYPK 3127GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCYPK 3128GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCHCYPK 3129GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYPK 3130GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCGCYPK 3131GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACFPK 3132GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACWPK 4920GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCACYPK 4921GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACTPK 4922GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACTPK 4923GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACTPK 4924GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACTPK 4925GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK 4926GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACTPK 4927GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACTPK 4928GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHC 4929GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHC 3133GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHC 3134GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHC 3135GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHC 3136GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHC 3137GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCHC 3138GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCAC 3139GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCAC 3140GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCAC 3141GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCAC 3142GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHC 3143GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCAC 3144GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCAC 3145GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCGCYGG 3146GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCYGG 3147GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCYGG 3148GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCYGG 3149GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCYGG 3150GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG 3151GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCYGG 3152GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYGG 3153GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACYGG 3154GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACYGG 3155GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACYGG 3156GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACYGG 3157GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG 3158GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACYGG 3159GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACYGG 3160GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYG 3161GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCGGG 3162GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCGGG 3163GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCGGG 3164GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCGGG 3165GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCGGG 3166GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCGGG 3167GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACFGG 3168GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACGGG 3169GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACGGG 3170GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACGGG 3171GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACGGG 3172GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCACGGG 3173GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACGGG 3174GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACGGG 3175GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACGG 3176GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYG 3177GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACGG 3178GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCTPK 3179GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCTPK 3180GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCTPK 3181GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK 3182GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCTPK 3183GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCHCTPK 3184GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCYPK 3185GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCYPK 3186GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCYPK 3187GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCYPK 3188GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYPK 3189GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCYPK 3190GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCHCYPK 3191GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACTPK 3192GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACTPK 3193GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACTPK 3194GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACTPK 3195GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK 3196GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACTPK 3197GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACTPK 3198GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHC 3199GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHC 3200GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHC 3201GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHC 3202GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHC 3203GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHC 3204GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCAC 3205GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCAC 3206GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCAC 3207GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCAC 3208GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHC 3209GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCAC 3210GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCAC 3211GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCHCYGG 3212GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCYGG 3213GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCHCYG 3214GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCACYG 3215GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCYGG 3216GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCYGG 3217GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCYGG 3218GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG 3219GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCYGG 3220GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCHCYGG 3221GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCACYG 3222GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACYGG 3223GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACYGG 3224GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACYGG 3225GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACYGG 3226GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG 3227GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACYGG 3228GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACYGG 3229GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCACFGG 3230GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCGGG 3231GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCGGG 3232GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCGGG 3233GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCGGG 3234GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCGGG 3235GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCGGG 3236GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACGGG 3237GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACGGG 3238GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACGGG 3239GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACGGG 3240GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCACTP 3241GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACTP 3242GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACTP 3243GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCTPK-amide 3244GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCTPK- 3245 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCTPK- 3246 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCTPK- 3247 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK- 3248 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCTPK- 3249 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCHCTPK- 3250 amideGVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCYPK-amide 3251GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCYPK- 3252 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCYPK- 3253 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCYPK- 3254 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCYPK- 3255 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCYPK- 3256 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCHCYPK- 3257 amideGVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACTPK-amide 3258GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACTPK- 3259 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACTPK- 3260 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACTPK- 3261 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCACTPK- 3262 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACTPK- 3263 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACTPK- 3264 amideGVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHC-amide 3265GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHC- 3266 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHC- 3267 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHC-amide 3268GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHC- 3269 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHC-amide 3270GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCAC-amide 3271GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCAC- 3272 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCAC- 3273 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCAC-amide 3274GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHC- 3275 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCAC- 3276 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCAC-amide 3277GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYGG-amide 3278GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCYGG-amide 3279GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCYGG- 3280 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCYGG- 3281 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCYGG- 3282 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG- 3283 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCYGG- 3284 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCFGG-amide 3285GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYG-amide 3286GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYG-amide 3287GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACYGG-amide 3288GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACYGG- 3289 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACYGG- 3290 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACYGG- 3291 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCACYGG- 3292 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACYGG- 3293 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACYGG- 3294 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYGG-amide 3295GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCGGG-amide 3296GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCGGG- 3297 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCGGG- 3298 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCGGG- 3299 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCGGG- 3300 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCGGG- 3301 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACGGG-amide 3302GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACFGG-amide 3303GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACGGG-amide 3304GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACGGG- 3305 amideGVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACGGG- 3306 amideGVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACGGG- 3307 amideGVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCACGGG- 3308 amideGVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACGGG- 3309 amideGVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACGGG- 3310 amideGVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCTPK-amide 3311GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCTPK- 3312 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCTPK- 3313 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCTPK- 3314 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK- 3315 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCTPK- 3316 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCHCTPK- 3317 amideGVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCYPK-amide 3318GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCYPK- 3319 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCYPK- 3320 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCYPK- 3321 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYPK- 3322 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCYPK- 3323 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCHCYPK- 3324 amideGVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACTPK-amide 3325GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACTPK- 3326 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACTPK- 3327 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACTPK- 3328 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCACTPK- 3329 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACTPK- 3330 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACTPK- 3331 amideGVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHC-amide 3332GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHC- 3333 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHC- 3334 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHC-amide 3335GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHC- 3336 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHC-amide 3337GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCAC-amide 3338GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCAC- 3339 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCAC- 3340 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCAC-amide 3341GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHC- 3342 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCAC-amide 3343GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCAC-amide 3344GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCHCWGG-amide 3345GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCYGG-amide 3346GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCYGG- 3347 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCYGG- 3348 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCYGG- 3349 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG- 3350 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCYGG- 3351 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCHCYGG- 3352 amideGVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCACYGG-amide 3353GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACYGG-amide 3354GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACYGG- 3355 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACYGG- 3356 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACYGG- 3357 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG- 3358 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACYGG- 3359 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACYGG- 3360 amideGVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCGGG-amide 3361GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCGGG- 3362 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCGGG- 3363 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCGGG- 3364 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCGGG- 3365 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCGGG- 3366 amideGVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACGGG-amide 3367GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACGGG- 3368 amideGVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACGGG- 3369 amideGVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACGGG- 3370 amideGVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCACTP- 3371 amideGVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACGGG- 3372 amideGVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACGGG- 3373 amideGVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMAGKCACYGG- 3374 amideGVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMAGKCACGGG- 3375 amideGVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMAGKCACY- 3376 amideAc-GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYGG- 3377 amideGVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMAGKCACYGG- 3378 amideGVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMAGKCHCYGG- 3379 amideGVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMAGKCACYGG 3380GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCACYGG-amide 3381GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCACYGG 3382GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCACY-amide 3383GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCHCYGG-amide 3384GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCHCYGG 3385GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCHCYPK 3386GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCAC 3387GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[1Nal]GG- 3388 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[1Nal]PK- 3389 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[2Nal]GG- 3390 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[Cha]GG- 3391 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[MePhe]GG- 3392 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[BiPhA]GG- 3393 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMAGKC[Aib]CYGG- 3394 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMAGKC[Abu]CYGG- 3395 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[1Nal] 3396GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCAC[1Nal]GG- 3397 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[4Bip]- 3398 amideGVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCAC[4Bip]GG- 3399 amideGVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCGGG 3400

TABLE 7F Addit6ional useful OSK1 peptide analogs: Ala27SubstitutedSeries SEQ ID Sequence/structure NO:GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK 3401GVIINVSCKISRQCLEPCKKAGMRFGACMNGKCHCTPK 3402GVIINVKCKISRQCLKPCKKAGMRFGACMNGKCHCTPK 3403GVIINVKCKISRQCLEPCKDAGMRFGACMNGKCHCTPK 3404GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3405GVIINVSCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3406GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK 3407GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYPK 3408Ac-GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK 3409GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK-amide 3410Ac-GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK- 3411 amideGVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYPK-amide 3412Ac-GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYPK 3413Ac-GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYPK- 3414 amideGVIINVKCKISRQCLKPCKKAGMRFGACMNGKCHCTPK-amide 3415Ac-GVIINVKCKISRQCLKPCKKAGMRFGACMNGKCHCTPK 3416Ac-GVIINVKCKISRQCLKPCKKAGMRFGACMNGKCHCTPK- 3417 amideAc-GVIINVKCKISRQCLEPCKDAGMRFGACMNGKCHCTPK 3418GVIINVKCKISRQCLEPCKDAGMRFGACMNGKCHCTPK-amide 3419Ac-GVIINVKCKISRQCLEPCKDAGMRFGACMNGKCHCTPK- 3420 amideGVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK-amide 3421Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK 3422Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK- 3423 amideGVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide 3424Ac-GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3425Ac-GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK- 3426 amideVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK 3427Ac-VIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK 3428VIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK-amide 3429Ac-VIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK- 3430 amideGVIINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK 3431Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK 3432GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK-amide 3433Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK- 3434 amideVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3435Ac-VIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3436VIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide 3437Ac-VIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK- 3438 amideNVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3439Ac-NVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3440NVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide 3441Ac-NVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide 3442KCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3443Ac-KCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3444KCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide 3445Ac-KCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide 3446CKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3447 Ac-CKISRQCLKPCKDAGMRFGACMNGKCHCTPK3448 CKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide 3449Ac-CKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide 3450GVIINVKCKISRQCLKPCKDAGMRNGACMNGKCHCTPK 3451GVIINVKCKISRQCLKPCKDAGMRNGACMNGKCHCTPK-amide 3452Ac-GVIINVKCKISRQCLKPCKDAGMRNGACMNGKCHCTPK 3453Ac-GVIINVKCKISRQCLKPCKDAGMRNGACMNGKCHCTPK- 3454 amideGVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK 3455GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-amide 3456Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK 3457Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK- 3458 amideGVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK 3459Ac-GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK 3460GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK-amide 3461Ac-GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK- 3462 amideTIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3463Ac-TIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3464TIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide 3465Ac-TIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK- 3466 amideGVKINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK 3467Ac-GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK 3468GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK-amide 3469Ac-GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK- 3470 amideGVKINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK 3471GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3472GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCACTPK 3473Ac-GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK 3474GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK-amide 3475Ac-GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK- 3476 amideAc-GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCACTPK 3477GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCACTPK-amide 3478Ac-GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCACTPK- 3479 amideAc-GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3480GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide 3481Ac-GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK- 3482 amideGVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCT 3483GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCTPK 3484GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCTPK 3485GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCTPK 3486GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCTPK 3487GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCTPK 3488GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCTPK 3489GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCHCTPK 3490GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCYPK 3491GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCYPK 3492GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCYPK 3493GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCYPK 3494GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCYPK 3495GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCYPK 3496GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCHCYPK 3497GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYPK 3498GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCGCYPK 3499GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACFPK 3500GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACWPK 3501GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCACYPK 3502GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACTPK 3503GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACTPK 3504GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACTPK 3505GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACTPK 3506GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCTPK 3507GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACTPK 3508GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACTPK 3509GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHC 3510GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHC 3511GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHC 3512GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHC 3513GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHC 3514GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHC 3515GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCHC 3516GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCAC 3517GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCAC 3518GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCAC 3519GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCAC 3520GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHC 3521GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCAC 3522GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCAC 3523GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCGCYGG 3524GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCYGG 3525GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCYGG 3526GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCYGG 3527GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCYGG 3528GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCYGG 3529GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCYGG 3530GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYGG 3531GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACYGG 3532GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACYGG 3533GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACYGG 3534GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACYGG 3535GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCYGG 3536GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACYGG 3537GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACYGG 3538GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYG 3539GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCGGG 3540GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCGGG 3541GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCGGG 3542GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCGGG 3543GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCGGG 3544GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCGGG 3545GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACFGG 3546GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACGGG 3547GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACGGG 3548GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACGGG 3549GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACGGG 3550GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCACGGG 3551GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACGGG 3552GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACGGG 3553GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACGG 3554GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYG 3555GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACGG 3556GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCTPK 3557GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCTPK 3558GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCTPK 3559GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCTPK 3560GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCTPK 3561GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCTPK 3562GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCYPK 3563GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCYPK 3564GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCYPK 3565GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCYPK 3566GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYPK 3567GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCYPK 3568GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCYPK 3569GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACTPK 3570GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACTPK 3571GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACTPK 3572GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACTPK 3573GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCTPK 3574GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACTPK 3575GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACTPK 3576GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHC 3577GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHC 3578GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHC 3579GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHC 3580GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHC 3581GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHC 3582GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCAC 3583GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCAC 3584GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCAC 3585GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCAC 3586GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHC 3587GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCAC 3588GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCAC 3589GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCHCYGG 3590GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCYGG 3591GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCHCYG 3592GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCACYG 3593GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCYGG 3594GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCYGG 3595GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCYGG 3596GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYGG 3597GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCYGG 3598GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCYGG 3599GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCACYG 3600GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACYGG 3601GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACYGG 3602GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACYGG 3603GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACYGG 3604GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYGG 3605GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACYGG 3606GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACYGG 3607GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCACFGG 3608GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCGGG 3609GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCGGG 3610GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCGGG 3611GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCGGG 3612GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCGGG 3613GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCGGG 3614GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACGGG 3615GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACGGG 3616GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACGGG 3617GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACGGG 3618GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCACTP 3619GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACTP 3620GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACTP 3621GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCTPK-amide 3622GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCTPK- 3623 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCTPK- 3624 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCTPK- 3625 amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCTPK- 3626 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCTPK- 3627 amideGVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCHCTPK- 3628 amideGVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCYPK-amide 3629GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCYPK- 3630 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCYPK- 3631 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCYPK- 3632 amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCYPK- 3633 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCYPK- 3634 amideGVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCHCYPK- 3635 amideGVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACTPK-amide 3636GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACTPK- 3637 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACTPK- 3638 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACTPK- 3639 amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCACTPK- 3640 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACTPK- 3641 amideGVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACTPK- 3642 amideGVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHC-amide 3643GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHC- 3644 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHC- 3645 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHC-amide 3646GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHC- 3647 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHC-amide 3648GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCAC-amide 3649GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCAC- 3650 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCAC- 3651 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCAC-amide 3652GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHC- 3653 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCAC-amide 3654GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCAC-amide 3655GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYGG-amide 3656GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCYGG-amide 3657GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCYGG- 3658 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCYGG- 3659 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCYGG- 3660 amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCYGG- 3661 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCYGG- 3662 amideGVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCFGG-amide 3663GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYG-amide 3664GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYG-amide 3665GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACYGG-amide 3666GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACYGG- 3667 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACYGG- 3668 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACYGG- 3669 amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCACYGG- 3670 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACYGG- 3671 amideGVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACYGG- 3672 amideGVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYGG-amide 3673GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCGGG-amide 3674GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCGGG- 3675 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCGGG- 3676 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCGGG- 3677 amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCGGG- 3678 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCGGG- 3679 amideGVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACGGG-amide 3680GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACFGG-amide 3681GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACGGG-amide 3682GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACGGG- 3683 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACGGG- 3684 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACGGG- 3685 amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCACGGG- 3686 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACGGG- 3687 amideGVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACGGG- 3688 amideGVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCTPK-amide 3689GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCTPK- 3690 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCTPK- 3691 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCTPK- 3692 amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCTPK- 3693 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCTPK- 3694 amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCTPK- 3695 amideGVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCYPK-amide 3696GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCYPK- 3697 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCYPK- 3698 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCYPK- 3699 amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYPK- 3700 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCYPK- 3701 amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCYPK- 3702 amideGVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACTPK-amide 3703GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACTPK- 3704 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACTPK- 3705 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACTPK- 3706 amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCACTPK- 3707 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACTPK- 3708 amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACTPK- 3709 amideGVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHC-amide 3710GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHC- 3711 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHC- 3712 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHC-amide 3713GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHC- 3714 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHC-amide 3715GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCAC-amide 3716GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCAC- 3717 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCAC- 3718 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCAC-amide 3719GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHC- 3720 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCAC-amide 3721GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCAC-amide 3722GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCHCWGG-amide 3723GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCYGG-amide 3724GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCYGG- 3725 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCYGG- 3726 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCYGG- 3727 amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYGG- 3728 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCYGG- 3729 amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCYGG- 3730 amideGVIINVKCKISRQCLKPCKEAGMRFGACMNGKCACYGG-amide 3731GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACYGG-amide 3732GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACYGG- 3733 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACYGG- 3734 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACYGG- 3735 amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYGG- 3736 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACYGG- 3737 amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACYGG- 3738 amideGVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCGGG-amide 3739GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCGGG- 3740 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCGGG- 3741 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCGGG- 3742 amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCGGG- 3743 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCGGG- 3744 amideGVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACGGG-amide 3745GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACGGG- 3746 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACGGG- 3747 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACGGG- 3748 amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCACTP- 3749 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACGGG- 3750 amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACGGG- 3751 amideGVIINVKCKISRQCLKPCK[Cpa]AGMRFGACMNGKCACYGG- 3752 amideGVIINVKCKISRQCLKPCK[Cpa]AGMRFGACMNGKCACGGG- 3753 amideGVIINVKCKISRQCLKPCK[Cpa]AGMRFGACMNGKCACY- 3754 amideAc-GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYGG- 3755 amideGVIINVKCKISRQCLKPCK[Aad]AGMRFGACMNGKCACYGG- 3756 amideGVIINVKCKISRQCLKPCK[Aad]AGMRFGACMNGKCHCYGG- 3757 amideGVIINVKCKISRQCLKPCK[Aad]AGMRFGACMNGKCACYGG 3758GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCACYGG-amide 3759GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCACYGG 3760GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCACY-amide 3761GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCHCYGG-amide 3762GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCHCYGG 3763GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCHCYPK 3764GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCAC 3765GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[1Nal]GG- 3766 amideGVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[1Nal]PK- 3767 amideGVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[2Nal]GG- 3768 amideGVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[Cha]GG- 3769 amideGVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[MePhe]GG- 3770 amideGVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[BiPhA]GG- 3771 amideGVIINVKCKISRQCLKPCKDAGMRFGACMNGKC[Aib]CYGG- 3772 amideGVIINVKCKISRQCLKPCKDAGMRFGACMNGKC[Abu]CYGG- 3773 amideGVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[1Nal] 3774GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCAC[1Nal]GG- 3775 amideGVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[4Bip]- 3776 amideGVIINVKCKISRQCLHPCKDAGMRFGACMNGKCAC[4Bip]GG- 3777 amideGVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCGGG 3778

TABLE 7G Additional useful OSK1 peptide analogs: Ala 29 SubstitutedSeries SEQ ID Sequence/structure NO:GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK 3779GVIINVSCKISRQCLEPCKKAGMRFGKCANGKCHCTPK 3780GVIINVKCKISRQCLKPCKKAGMRFGKCANGKCHCTPK 3781GVIINVKCKISRQCLEPCKDAGMRFGKCANGKCHCTPK 3782GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3783GVIINVSCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3784GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK 3785GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYPK 3786Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK 3787GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK-amide 3788Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK- 3789 amideGVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYPK-amide 3790Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYPK 3791Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYPK- 3792 amideGVIINVKCKISRQCLKPCKKAGMRFGKCANGKCHCTPK-amide 3793Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCANGKCHCTPK 3794Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCANGKCHCTPK- 3795 amideAc-GVIINVKCKISRQCLEPCKDAGMRFGKCANGKCHCTPK 3796GVIINVKCKISRQCLEPCKDAGMRFGKCANGKCHCTPK-amide 3797Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCANGKCHCTPK- 3798 amideGVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK-amide 3799Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK 3800Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK- 3801 amideGVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide 3802Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3803Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK- 3804 amideVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK 3805Ac-VIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK 3806VIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK-amide 3807Ac-VIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK- 3808 amideGVIINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK 3809Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK 3810GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK-amide 3811Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK- 3812 amideVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3813Ac-VIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3814VIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide 3815Ac-VIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK- 3816 amideNVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3817Ac-NVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3818NVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide 3819Ac-NVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide 3820KCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3821Ac-KCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3822KCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide 3823Ac-KCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide 3824CKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3825 Ac-CKISRQCLKPCKDAGMRFGKCANGKCHCTPK3826 CKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide 3827Ac-CKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide 3828GVIINVKCKISRQCLKPCKDAGMRNGKCANGKCHCTPK 3829GVIINVKCKISRQCLKPCKDAGMRNGKCANGKCHCTPK-amide 3830Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCANGKCHCTPK 3831Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCANGKCHCTPK- 3832 amideGVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK 3833GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-amide 3834Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK 3835Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK- 3836 amideGVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK 3837Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK 3838GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK-amide 3839Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK- 3840 amideTIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3841Ac-TIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3842TIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide 3843Ac-TIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK- 3844 amideGVKINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK 3845Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK 3846GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK-amide 3847Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK- 3848 amideGVKINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK 3849GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3850GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCACTPK 3851Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK 3852GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK-amide 3853Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK- 3854 amideAc-GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCACTPK 3855GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCACTPK-amide 3856Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCACTPK- 3857 amideAc-GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3858GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide 3859Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK- 3860 amideGVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCT 3861GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCTPK 3862GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCTPK 3863GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCTPK 3864GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCTPK 3865GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCTPK 3866GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCTPK 3867GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCHCTPK 3868GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCYPK 3869GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCYPK 3870GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCYPK 3871GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCYPK 3872GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCYPK 3873GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCYPK 3874GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCHCYPK 3875GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYPK 3876GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCGCYPK 3877GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACFPK 3878GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACWPK 3879GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCACYPK 3880GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACTPK 3881GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACTPK 3882GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACTPK 3883GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACTPK 3884GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCTPK 3885GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACTPK 3886GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACTPK 3887GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHC 3888GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHC 3889GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHC 3890GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHC 3891GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHC 3892GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHC 3893GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCHC 3894GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCAC 3895GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCAC 3896GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCAC 3897GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCAC 3898GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHC 3899GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCAC 3900GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCAC 3901GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCGCYGG 3902GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCYGG 3903GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCYGG 3904GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCYGG 3905GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCYGG 3906GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCYGG 3907GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCYGG 3908GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYGG 3909GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACYGG 3910GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACYGG 3911GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACYGG 3912GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACYGG 3913GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCYGG 3914GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACYGG 3915GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACYGG 3916GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYG 3917GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCGGG 3918GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCGGG 3919GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCGGG 3920GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCGGG 3921GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCGGG 3922GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCGGG 3923GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACFGG 3924GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACGGG 3925GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACGGG 3926GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACGGG 3927GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACGGG 3928GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCACGGG 3929GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACGGG 3930GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACGGG 3931GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACGG 3932GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYG 3933GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACGG 3934GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCTPK 3935GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCTPK 3936GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCTPK 3937GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCTPK 3938GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCTPK 3939GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCTPK 3940GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCYPK 3941GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCYPK 3942GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCYPK 3943GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCYPK 3944GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYPK 3945GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCYPK 3946GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCYPK 3947GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACTPK 3948GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACTPK 3949GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACTPK 3950GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACTPK 3951GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCTPK 3952GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACTPK 3953GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACTPK 3954GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHC 3955GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHC 3956GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHC 3957GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHC 3958GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHC 3959GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHC 3960GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCAC 3961GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCAC 3962GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCAC 3963GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCAC 3964GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHC 3965GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCAC 3966GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCAC 3967GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCHCYGG 3968GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCYGG 3969GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCHCYG 3970GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCACYG 3971GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCYGG 3972GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCYGG 3973GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCYGG 3974GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYGG 3975GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCYGG 3976GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCYGG 3977GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCACYG 3978GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACYGG 3979GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACYGG 3980GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACYGG 3981GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACYGG 3982GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYGG 3983GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACYGG 3984GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACYGG 3985GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCACFGG 3986GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCGGG 3987GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCGGG 3988GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCGGG 3989GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCGGG 3990GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCGGG 3991GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCGGG 3992GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACGGG 3993GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACGGG 3994GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACGGG 3995GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACGGG 3996GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCACTP 3997GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACTP 3998GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACTP 3999GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCTPK-amide 4000GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCTPK- 4001 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCTPK- 4002 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCTPK- 4003 amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCTPK- 4004 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCTPK- 4005 amideGVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCHCTPK- 4006 amideGVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCYPK-amide 4007GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCYPK- 4008 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCYPK- 4009 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCYPK- 4010 amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCYPK- 4011 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCYPK- 4012 amideGVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCHCYPK- 4013 amideGVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACTPK-amide 4014GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACTPK- 4015 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACTPK- 4016 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACTPK- 4017 amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCACTPK- 4018 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACTPK- 4019 amideGVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACTPK- 4020 amideGVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHC-amide 4021GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHC- 4022 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHC- 4023 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHC-amide 4024GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHC- 4025 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHC-amide 4026GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCAC-amide 4027GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCAC- 4028 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCAC- 4029 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCAC-amide 4030GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHC- 4031 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCAC-amide 4032GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCAC-amide 4033GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYGG-amide 4034GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCYGG-amide 4035GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCYGG- 4036 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCYGG- 4037 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCYGG- 4038 amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCYGG- 4039 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCYGG- 4040 amideGVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCFGG-amide 4041GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYG-amide 4042GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYG-amide 4043GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACYGG-amide 4044GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACYGG- 4045 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACYGG- 4046 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACYGG- 4047 amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCACYGG- 4048 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACYGG- 4049 amideGVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACYGG- 4050 amideGVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYGG-amide 4051GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCGGG-amide 4052GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCGGG- 4053 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCGGG- 4054 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCGGG- 4055 amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCGGG- 4056 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCGGG- 4057 amideGVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACGGG-amide 4058GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACFGG-amide 4059GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACGGG-amide 4060GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACGGG- 4061 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACGGG- 4062 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACGGG- 4063 amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCACGGG- 4064 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACGGG- 4065 amideGVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACGGG- 4066 amideGVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCTPK-amide 4067GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCTPK- 4068 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCTPK- 4069 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCTPK- 4070 amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCTPK- 4071 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCTPK- 4072 amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCTPK- 4073 amideGVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCYPK-amide 4074GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCYPK- 4075 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCYPK- 4076 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCYPK- 4077 amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYPK- 4078 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCYPK- 4079 amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCYPK- 4080 amideGVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACTPK-amide 4081GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACTPK- 4082 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACTPK- 4083 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACTPK- 4084 amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCACTPK- 4085 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACTPK- 4086 amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACTPK- 4087 amideGVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHC-amide 4088GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHC- 4089 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHC- 4090 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHC-amide 4091GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHC- 4092 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHC-amide 4093GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCAC-amide 4094GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCAC- 4095 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCAC- 4096 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCAC-amide 4097GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHC- 4098 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCAC-amide 4099GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCAC-amide 4100GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCHCWGG-amide 4101GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCYGG-amide 4102GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCYGG- 4103 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCYGG- 4104 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCYGG- 4105 amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYGG- 4106 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCYGG- 4107 amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCYGG- 4108 amideGVIINVKCKISRQCLKPCKEAGMRFGKCANGKCACYGG-amide 4109GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACYGG-amide 4110GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACYGG- 4111 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACYGG- 4112 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACYGG- 4113 amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYGG- 4114 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACYGG- 4115 amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACYGG- 4116 amideGVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCGGG-amide 4117GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCGGG- 4118 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCGGG- 4119 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCGGG- 4120 amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCGGG- 4121 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCGGG- 4122 amideGVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACGGG-amide 4123GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACGGG- 4124 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACGGG- 4125 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACGGG- 4126 amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCACTP- 4127 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACGGG- 4128 amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACGGG- 4129 amideGVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCANGKCACYGG- 4130 amideGVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCANGKCACGGG- 4131 amideGVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCANGKCACY- 4132 amideAc-GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYGG- 4133 amideGVIINVKCKISRQCLKPCK[Aad]AGMRFGKCANGKCACYGG- 4134 amideGVIINVKCKISRQCLKPCK[Aad]AGMRFGKCANGKCHCYGG- 4135 amideGVIINVKCKISRQCLKPCK[Aad]AGMRFGKCANGKCACYGG 4136GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCACYGG-amide 4137GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCACYGG 4138GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCACY-amide 4139GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCHCYGG-amide 4140GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCHCYGG 4141GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCHCYPK 4142GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCAC 4143GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[1Nal]GG- 4144 amideGVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[1Nal]PK- 4145 amideGVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[2Nal]GG- 4146 amideGVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[Cha]GG- 4147 amideGVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[MePhe]GG- 4148 amideGVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[BiPhA]GG- 4149 amideGVIINVKCKISRQCLKPCKDAGMRFGKCANGKC[Aib]CYGG- 4150 amideGVIINVKCKISRQCLKPCKDAGMRFGKCANGKC[Abu]CYGG- 4151 amideGVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[1Nal] 4152GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCAC[1Nal]GG- 4153 amideGVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[4Bip]- 4154 amideGVIINVKCKISRQCLHPCKDAGMRFGKCANGKCAC[4Bip]GG- 4155 amideGVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCGGG 4156

TABLE 7H Additional useful OSK1 peptide analogs: Ala 30 SubstitutedSeries SEQ ID Sequence/structure NO:GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK 4157GVIINVSCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK 4158GVIINVKCKISRQCLKPCKKAGMRFGKCMAGKCHCTPK 4159GVIINVKCKISRQCLEPCKDAGMRFGKCMAGKCHCTPK 4160GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4161GVIINVSCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4162GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK 4163GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYPK 4164Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK 4165GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 4166Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK- 4167 amideGVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYPK-amide 4168Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYPK 4169Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYPK- 4170 amideGVIINVKCKISRQCLKPCKKAGMRFGKCMAGKCHCTPK-amide 4171Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCMAGKCHCTPK 4172Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCMAGKCHCTPK- 4173 amideAc-GVIINVKCKISRQCLEPCKDAGMRFGKCMAGKCHCTPK 4174GVIINVKCKISRQCLEPCKDAGMRFGKCMAGKCHCTPK-amide 4175Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCMAGKCHCTPK- 4176 amideGVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK-amide 4177Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK 4178Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK- 4179 amideGVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 4180Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4181Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK- 4182 amideVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK 4183Ac-VIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK 4184VIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK-amide 4185Ac-VIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK- 4186 amideGVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK 4187Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK 4188GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK-amide 4189Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK- 4190 amideVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4191Ac-VIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4192VIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 4193Ac-VIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK- 4194 amideNVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4195Ac-NVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4196NVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 4197Ac-NVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 4198KCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4199Ac-KCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4200KCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 4201Ac-KCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 4202CKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4203 Ac-CKISRQCLKPCKDAGMRFGKCMAGKCHCTPK4204 CKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 4205Ac-CKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 4206GVIINVKCKISRQCLKPCKDAGMRNGKCMAGKCHCTPK 4207GVIINVKCKISRQCLKPCKDAGMRNGKCMAGKCHCTPK-amide 4208Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCMAGKCHCTPK 4209Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCMAGKCHCTPK- 4210 amideGVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK 4211GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-amide 4212Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK 4213Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK- 4214 amideGVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK 4215Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK 4216GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK-amide 4217Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK- 4218 amideTIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4219Ac-TIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4220TIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 4221Ac-TIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK- 4222 amideGVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK 4223Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK 4224GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK-amide 4225Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK- 4226 amideGVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK 4227GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4228GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCACTPK 4229Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK 4230GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK-amide 4231Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK- 4232 amideAc-GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCACTPK 4233GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCACTPK-amide 4234Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCACTPK- 4235 amideAc-GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4236GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 4237Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK- 4238 amideGVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCT 4239GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCTPK 4240GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCTPK 4241GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCTPK 4242GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCTPK 4243GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK 4244GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCTPK 4245GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCHCTPK 4246GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCYPK 4247GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCYPK 4248GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCYPK 4249GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCYPK 4250GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCYPK 4251GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCYPK 4252GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCHCYPK 4253GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYPK 4254GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCGCYPK 4255GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACFPK 4256GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACWPK 4257GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCACYPK 4258GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACTPK 4259GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCACTPK 4260GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACTPK 4261GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACTPK 4262GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK 4263GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACTPK 4264GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACTPK 4265GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHC 4266GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHC 4267GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHC 4268GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHC 4269GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHC 4270GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHC 4271GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCHC 4272GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCAC 4273GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCAC 4274GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCAC 4275GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCAC 4276GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHC 4277GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCAC 4278GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCAC 4279GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCGCYGG 4280GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCYGG 4281GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCYGG 4282GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCYGG 4283GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCYGG 4284GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG 4285GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCYGG 4286GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYGG 4287GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACYGG 4288GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCACYGG 4289GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACYGG 4290GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACYGG 4291GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG 4292GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACYGG 4293GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACYGG 4294GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYG 4295GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCGGG 4296GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCGGG 4297GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCGGG 4298GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCGGG 4299GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCGGG 4300GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCGGG 4301GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACFGG 4302GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACGGG 4303GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCACGGG 4304GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACGGG 4305GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACGGG 4306GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCACGGG 4307GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACGGG 4308GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACGGG 4309GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACGG 4310GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYG 4311GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACGG 4312GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCTPK 4313GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCTPK 4314GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCTPK 4315GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK 4316GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCTPK 4317GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCTPK 4318GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCYPK 4319GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCYPK 4320GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCYPK 4321GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCYPK 4322GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYPK 4323GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCYPK 4324GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCYPK 4325GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACTPK 4326GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACTPK 4327GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACTPK 4328GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACTPK 4329GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK 4330GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACTPK 4331GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACTPK 4332GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHC 4333GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHC 4334GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHC 4335GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHC 4336GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHC 4337GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHC 4338GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCAC 4339GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCAC 4340GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCAC 4341GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCAC 4342GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHC 4343GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCAC 4344GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCAC 4345GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCHCYGG 4346GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCYGG 4347GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCHCYG 4348GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCACYG 4349GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCYGG 4350GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCYGG 4351GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCYGG 4352GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG 4353GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCYGG 4354GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCYGG 4355GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCACYG 4356GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACYGG 4357GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACYGG 4358GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACYGG 4359GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACYGG 4360GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG 4361GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACYGG 4362GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACYGG 4363GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCACFGG 4364GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCGGG 4365GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCGGG 4366GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCGGG 4367GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCGGG 4368GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCGGG 4369GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCGGG 4370GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACGGG 4371GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACGGG 4372GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACGGG 4373GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACGGG 4374GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCACTP 4375GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACTP 4376GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACTP 4377GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCTPK-amide 4378GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCTPK- 4379 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCTPK- 4380 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCTPK- 4381 amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK- 4382 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCTPK- 4383 amideGVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCHCTPK- 4384 amideGVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCYPK-amide 4385GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCYPK- 4386 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCYPK- 4387 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCYPK- 4388 amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCYPK- 4389 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCYPK- 4390 amideGVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCHCYPK- 4391 amideGVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACTPK-amide 4392GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCACTPK- 4393 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACTPK- 4394 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACTPK- 4395 amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCACTPK- 4396 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACTPK- 4397 amideGVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACTPK- 4398 amideGVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHC-amide 4399GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHC- 4400 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHC- 4401 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHC-amide 4402GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHC- 4403 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHC-amide 4404GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCAC-amide 4405GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCAC- 4406 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCAC- 4407 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCAC-amide 4408GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHC- 4409 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCAC-amide 4410GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCAC-amide 4411GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYGG-amide 4412GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCYGG-amide 4413GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCYGG- 4414 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCYGG- 4415 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCYGG- 4416 amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG- 4417 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCYGG- 4418 amideGVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCFGG-amide 4419GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYG-amide 4420GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYG-amide 4421GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACYGG-amide 4422GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCACYGG- 4423 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACYGG- 4424 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACYGG- 4425 amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCACYGG- 4426 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACYGG- 4427 amideGVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACYGG- 4428 amideGVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYGG-amide 4429GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCGGG-amide 4430GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCGGG- 4431 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCGGG- 4432 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCGGG- 4433 amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCGGG- 4434 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCGGG- 4435 amideGVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACGGG-amide 4436GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACFGG-amide 4437GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACGGG-amide 4438GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCACGGG- 4439 amideGVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACGGG- 4440 amideGVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACGGG- 4441 amideGVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCACGGG- 4442 amideGVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACGGG- 4443 amideGVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACGGG- 4444 amideGVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCTPK-amide 4445GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCTPK- 4446 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCTPK- 4447 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCTPK- 4448 amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK- 4449 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCTPK- 4450 amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCTPK- 4451 amideGVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCYPK-amide 4452GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCYPK- 4453 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCYPK- 4454 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCYPK- 4455 amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYPK- 4456 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCYPK- 4457 amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCYPK- 4458 amideGVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACTPK-amide 4459GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACTPK- 4460 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACTPK- 4461 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACTPK- 4462 amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCACTPK- 4463 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACTPK- 4464 amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACTPK- 4465 amideGVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHC-amide 4466GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHC- 4467 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHC- 4468 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHC-amide 4469GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHC- 4470 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHC-amide 4471GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCAC-amide 4472GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCAC- 4473 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCAC- 4474 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCAC-amide 4475GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHC- 4476 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCAC-amide 4477GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCAC-amide 4478GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCHCWGG-amide 4479GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCYGG-amide 4480GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCYGG- 4481 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCYGG- 4482 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCYGG- 4483 amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG- 4484 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCYGG- 4485 amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCYGG- 4486 amideGVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCACYGG-amide 4487GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACYGG-amide 4488GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACYGG- 4489 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACYGG- 4490 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACYGG- 4491 amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG- 4492 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACYGG- 4493 amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACYGG- 4494 amideGVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCGGG-amide 4495GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCGGG- 4496 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCGGG- 4497 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCGGG- 4498 amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCGGG- 4499 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCGGG- 4500 amideGVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACGGG-amide 4501GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACGGG- 4502 amideGVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACGGG- 4503 amideGVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACGGG- 4504 amideGVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCACTP- 4505 amideGVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACGGG- 4506 amideGVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACGGG- 4507 amideGVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMAGKCACYGG- 4508 amideGVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMAGKCACGGG- 4509 amideGVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMAGKCACY- 4510 amideAc-GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYGG- 4511 amideGVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMAGKCACYGG- 4512 amideGVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMAGKCHCYGG- 4513 amideGVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMAGKCACYGG 4514GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCACYGG-amide 4515GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCACYGG 4516GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCACY-amide 4517GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCHCYGG-amide 4518GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCHCYGG 4519GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCHCYPK 4520GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCAC 4521GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[1Nal]GG- 4522 amideGVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[1Nal]PK- 4523 amideGVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[2Nal]GG- 4524 amideGVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[Cha]GG- 4525 amideGVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[MePhe]GG- 4526 amideGVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[BiPhA]GG- 4527 amideGVIINVKCKISRQCLKPCKDAGMRFGKCMAGKC[Aib]CYGG- 4528 amideGVIINVKCKISRQCLKPCKDAGMRFGKCMAGKC[Abu]CYGG- 4529 amideGVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[1Nal] 4530GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCAC[1Nal]GG- 4531 amideGVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[4Bip]- 4532 amideGVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCAC[4Bip]GG- 4533 amideGVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCGGG 4534

TABLE 7I Additional useful OSK1 peptide analogs: CombinedAla-11, 12, 27, 29, 30 Substituted Series SEQ ID Sequence/structure NO:GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK 4535GVIINVSCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK 4536GVIINVKCKIAAQCLKPCKKAGMRFGACAAGKCHCTPK 4537GVIINVKCKIAAQCLEPCKDAGMRFGACAAGKCHCTPK 4538GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4539GVIINVSCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4540GVIINVKCKISPQCLKPCKDAGMRFGACAAGKCHCTPK 4541GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYPK 4542Ac-GVIINVKCKISPQCLKPCKDAGMRFGACAAGKCHCTPK 4543GVIINVKCKISPQCLKPCKDAGMRFGACAAGKCHCTPK-amide 4544Ac-GVIINVKCKISPQCLKPCKDAGMRFGACAAGKCHCTPK- 4545 amideGVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYPK-amide 4546Ac-GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYPK 4547Ac-GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYPK- 4548 amideGVIINVKCKIAAQCLKPCKKAGMRFGACAAGKCHCTPK-amide 4549Ac-GVIINVKCKIAAQCLKPCKKAGMRFGACAAGKCHCTPK 4550Ac-GVIINVKCKIAAQCLKPCKKAGMRFGACAAGKCHCTPK- 4551 amideAc-GVIINVKCKIAAQCLEPCKDAGMRFGACAAGKCHCTPK 4552GVIINVKCKIAAQCLEPCKDAGMRFGACAAGKCHCTPK-amide 4553Ac-GVIINVKCKIAAQCLEPCKDAGMRFGACAAGKCHCTPK- 4554 amideGVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK-amide 4555Ac-GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK 4556Ac-GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK- 4557 amideGVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide 4558Ac-GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4559Ac-GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK- 4560 amideVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK 4561Ac-VIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK 4562VIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK-amide 4563Ac-VIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK- 4564 amideGVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK 4565Ac-GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK 4566GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK-amide 4567Ac-GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK- 4568 amideVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4569Ac-VIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4570VIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide 4571Ac-VIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK- 4572 amideNVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4573Ac-NVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4574NVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide 4575Ac-NVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide 4576KCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4577Ac-KCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4578KCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide 4579Ac-KCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide 4580CKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4581 Ac-CKIAAQCLKPCKDAGMRFGACAAGKCHCTPK4582 CKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide 4583Ac-CKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide 4584GVIINVKCKIAAQCLKPCKDAGMRNGACAAGKCHCTPK 4585GVIINVKCKIAAQCLKPCKDAGMRNGACAAGKCHCTPK-amide 4586Ac-GVIINVKCKIAAQCLKPCKDAGMRNGACAAGKCHCTPK 4587Ac-GVIINVKCKIAAQCLKPCKDAGMRNGACAAGKCHCTPK- 4588 amideGVIINVKCKIAAQCLKPCKDAGMRFGKCMNRKCHCTPK 4589GVIINVKCKIAAQCLKPCKDAGMRFGKCMNRKCHCTPK-amide 4590Ac-GVIINVKCKIAAQCLKPCKDAGMRFGKCMNRKCHCTPK 4591Ac-GVIINVKCKIAAQCLKPCKDAGMRFGKCMNRKCHCTPK- 4592 amideGVIINVKCKISKQCLKPCRDAGMRFGACAAGKCHCTPK 4593Ac-GVIINVKCKISKQCLKPCRDAGMRFGACAAGKCHCTPK 4594GVIINVKCKISKQCLKPCRDAGMRFGACAAGKCHCTPK-amide 4595Ac-GVIINVKCKISKQCLKPCRDAGMRFGACAAGKCHCTPK- 4596 amideTIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4597Ac-TIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4598TIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide 4599Ac-TIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK- 4600 amideGVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK 4601Ac-GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK 4602GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK-amide 4603Ac-GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK- 4604 amideGVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK 4605GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4606GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCACTPK 4607Ac-GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK 4608GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK-amide 4609Ac-GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK- 4610 amideAc-GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCACTPK 4611GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCACTPK-amide 4612Ac-GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCACTPK- 4613 amideAc-GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4614GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide 4615Ac-GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK- 4616 amideGVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCT 4617GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCTPK 4618GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCTPK 4619GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCTPK 4620GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCTPK 4621GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCTPK 4622GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCTPK 4623GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCHCTPK 4624GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCYPK 4625GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCYPK 4626GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCYPK 4627GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCYPK 4628GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCYPK 4629GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCYPK 4630GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCHCYPK 4631GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYPK 4632GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCGCYPK 4633GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACFPK 4634GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACWPK 4635GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCACYPK 4636GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACTPK 4637GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACTPK 4638GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACTPK 4639GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACTPK 4640GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCTPK 4641GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACTPK 4642GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACTPK 4643GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHC 4644GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHC 4645GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHC 4646GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHC 4647GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHC 4648GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHC 4649GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCHC 4650GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCAC 4651GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCAC 4652GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCAC 4653GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCAC 4654GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHC 4655GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCAC 4656GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCAC 4657GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCGCYGG 4658GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCYGG 4659GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCYGG 4660GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCYGG 4661GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCYGG 4662GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCYGG 4663GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCYGG 4664GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYGG 4665GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACYGG 4666GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACYGG 4667GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACYGG 4668GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACYGG 4669GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCYGG 4670GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACYGG 4671GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACYGG 4672GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYG 4673GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCGGG 4674GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCGGG 4675GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCGGG 4676GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCGGG 4677GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCGGG 4678GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCGGG 4679GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACFGG 4680GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACGGG 4681GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACGGG 4682GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACGGG 4683GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACGGG 4684GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCACGGG 4685GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACGGG 4686GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACGGG 4687GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACGG 4688GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYG 4689GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACGG 4690GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCTPK 4691GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCTPK 4692GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCTPK 4693GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCTPK 4694GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCTPK 4695GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCTPK 4696GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCYPK 4697GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCYPK 4698GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCYPK 4699GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCYPK 4700GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYPK 4701GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCYPK 4702GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCYPK 4703GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACTPK 4704GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACTPK 4705GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACTPK 4706GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACTPK 4707GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCTPK 4708GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACTPK 4709GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCACTPK 4710GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHC 4711GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHC 4712GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHC 4713GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHC 4714GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHC 4715GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHC 4716GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCAC 4717GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCAC 4718GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCAC 4719GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCAC 4720GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHC 4721GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCAC 4722GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCAC 4723GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCHCYGG 4724GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCYGG 4725GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCHCYG 4726GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCACYG 4727GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCYGG 4728GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCYGG 4729GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCYGG 4730GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYGG 4731GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCYGG 4732GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCYGG 4733GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCACYG 4734GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACYGG 4735GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACYGG 4736GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACYGG 4737GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACYGG 4738GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYGG 4739GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACYGG 4740GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCACYGG 4741GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCACFGG 4742GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCGGG 4743GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCGGG 4744GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCGGG 4745GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCGGG 4746GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCGGG 4747GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCGGG 4748GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACGGG 4749GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACGGG 4750GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACGGG 4751GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACGGG 4752GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCACTP 4753GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACTP 4754GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCACTP 4755GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCTPK-amide 4756GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCTPK- 4757 amideGVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCTPK- 4758 amideGVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCTPK- 4759 amideGVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCTPK- 4760 amideGVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCTPK- 4761 amideGVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCHCTPK- 4762 amideGVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCYPK-amide 4763GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCYPK- 4764 amideGVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCYPK- 4765 amideGVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCYPK- 4766 amideGVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCYPK- 4767 amideGVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCYPK- 4768 amideGVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCHCYPK- 4769 amideGVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACTPK-amide 4770GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACTPK- 4771 amideGVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACTPK- 4772 amideGVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACTPK- 4773 amideGVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCACTPK- 4774 amideGVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACTPK- 4775 amideGVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACTPK- 4776 amideGVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHC-amide 4777GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHC- 4778 amideGVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHC- 4779 amideGVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHC-amide 4780GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHC- 4781 amideGVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHC- 4782 amideGVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCAC-amide 4783GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCAC- 4784 amideGVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCAC- 4785 amideGVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCAC-amide 4786GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHC- 4787 amideGVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCAC-amide 4788GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCAC-amide 4789GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYGG-amide 4790GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCYGG-amide 4791GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCYGG- 4792 amideGVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCYGG- 4793 amideGVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCYGG- 4794 amideGVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCYGG- 4795 amideGVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCYGG- 4796 amideGVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCFGG-amide 4797GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYG-amide 4798GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYG-amide 4799GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACYGG-amide 4800GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACYGG- 4801 amideGVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACYGG- 4802 amideGVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACYGG- 4803 amideGVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCACYGG- 4804 amideGVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACYGG- 4805 amideGVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACYGG- 4806 amideGVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYGG-amide 4807GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCGGG-amide 4808GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCGGG- 4809 amideGVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCGGG- 4810 amideGVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCGGG- 4811 amideGVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCGGG- 4812 amideGVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCGGG- 4813 amideGVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACGGG-amide 4814GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACFGG-amide 4815GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACGGG-amide 4816GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACGGG- 4817 amideGVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACGGG- 4818 amideGVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACGGG- 4819 amideGVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCACGGG- 4820 amideGVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACGGG- 4821 amideGVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACGGG- 4822 amideGVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCTPK-amide 4823GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCTPK- 4824 amideGVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCTPK- 4825 amideGVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCTPK- 4826 amideGVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCTPK- 4827 amideGVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCTPK- 4828 amideGVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCTPK- 4829 amideGVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCYPK-amide 4830GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCYPK- 4831 amideGVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCYPK- 4832 amideGVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCYPK- 4833 amideGVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYPK- 4834 amideGVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCYPK- 4835 amideGVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCYPK- 4836 amideGVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACTPK-amide 4837GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACTPK- 4838 amideGVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACTPK- 4839 amideGVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACTPK- 4840 amideGVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCACTPK- 4841 amideGVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACTPK- 4842 amideGVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCACTPK- 4843 amideGVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHC-amide 4844GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHC- 4845 amideGVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHC- 4846 amideGVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHC-amide 4847GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHC- 4848 amideGVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHC-amide 4849GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCAC-amide 4850GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCAC- 4851 amideGVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCAC- 4852 amideGVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCAC-amide 4853GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHC- 4854 amideGVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCAC-amide 4855GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCAC-amide 4856GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCHCWGG-amide 4857GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCYGG-amide 4858GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCYGG- 4859 amideGVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCYGG- 4860 amideGVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCYGG- 4861 amideGVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYGG- 4862 amideGVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCYGG- 4863 amideGVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCYGG- 4864 amideGVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCACYGG-amide 4865GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACYGG-amide 4866GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACYGG- 4867 amideGVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACYGG- 4868 amideGVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACYGG- 4869 amideGVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYGG- 4870 amideGVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACYGG- 4871 amideGVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCACYGG- 4872 amideGVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCGGG-amide 4873GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCGGG- 4874 amideGVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCGGG- 4875 amideGVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCGGG- 4876 amideGVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCGGG- 4877 amideGVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCGGG- 4878 amideGVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACGGG-amide 4879GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACGGG- 4880 amideGVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACGGG- 4881 amideGVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACGGG- 4882 amideGVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCACTP- 4883 amideGVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACGGG- 4884 amideGVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCACGGG- 4885 amideGVIINVKCKIAAQCLKPCK[Cpa]AGMRFGACAAGKCACYGG- 4886 amideGVIINVKCKIAAQCLKPCK[Cpa]AGMRFGACAAGKCACGGG- 4887 amideGVIINVKCKIAAQCLKPCK[Cpa]AGMRFGACAAGKCACY- 4888 amideAc-GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYGG- 4889 amideGVIINVKCKIAAQCLKPCK[Aad]AGMRFGACAAGKCACYGG- 4890 amideGVIINVKCKIAAQCLKPCK[Aad]AGMRFGACAAGKCHCYGG- 4891 amideGVIINVKCKIAAQCLKPCK[Aad]AGMRFGACAAGKCACYGG 4892GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCACYGG-amide 4893GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCACYGG 4894GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCACY-amide 4895GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCHCYGG-amide 4896GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCHCYGG 4897GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCHCYPK 4898GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCAC 4899GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[1Nal]GG- 4900 amideGVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[1Nal]PK- 4901 amideGVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[2Nal]GG- 4902 amideGVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[Cha]GG- 4903 amideGVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[MePhe]GG- 4904 amideGVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[BiPhA]GG- 4905 amideGVIINVKCKIAAQCLKPCKDAGMRFGACAAGKC[Aib]CYGG- 4906 amideGVIINVKCKIAAQCLKPCKDAGMRFGACAAGKC[Abu]CYGG- 4907 amideGVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[1Nal] 4908GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCAC[1Nal]GG- 4909 amideGVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[4Bip]- 4910 amideGVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCAC[4Bip]GG- 4911 amideGVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCGGG 4912GIINVKCKISAQCLKPCRDAGMRFGKCMNGKCACTPK 4916

TABLE 7J Additional useful OSK1 peptide analogs SEQ Short-hand IDSequence/Structure designation NO: GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCGC[Gly34]OSK1 4930 TPK GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCSC [Ser34]OSK14931 TPK GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCTC [Thr34]OSK1 4932 TPKGVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCNC [Asn34]OSK1 4933 TPKGVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCVC [Val34]OSK1 4934 TPKGVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCLC [Leu34]OSK1 4935 TPKGVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCIC [Ile34]OSK1 4936 TPKGVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCPC [Pro34]OSK1 4937 TPKGVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCMC [Met34]OSK1 4938 TPKGVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCQC [Gln34]OSK1 4939 TPKGVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCKC [Lys34]OSK1 4940 TPKGVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCDC [Asp34]OSK1 4941 TPKWVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC [Trp1]OSK1 4942 WPKGVWINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC [Trp3]OSK1 4943 TPKGVIIWVKCKISRQCLEPCKKAGMRFGKCMNGKCHC [Trp5]OSK1 4944 TPK[1Nal]VIINVKCKISRQCLEPCKKAGMRFGKCMN [1Nal1]OSK1 4945 GKCHCWPKGV[1Nal]INVKCKISRQCLEPCKKAGMRFGKCMN [1Nal3]OSK1 4946 GKCHCTPKGVII[1Nal]VKCKISRQCLEPCKKAGMRFGKCMN [1Nal5]OSK1 4947 GKCHCTPKGVIKNVKCKISRQCLEPCKKAGMRFGKCMNGKCHC [Lys4]OSK1 4948 TPKGVIKNVKCKISRQCLEPCKKAGMRFGKCMNGKCAC [Lys4, Ala34]OSK1 4949 TPK[1Nal]VIINVKCKISRQCLEPCKKAGMRFGKCMN [1Nal1; 4950 GKCACWPK Ala34]OSK1GV[1Nal]INVKCKISRQCLEPCKKAGMRFGKCMN [1Nal3; 4951 GKCACTPK Ala34]OSK1GVII[1Nal]VKCKISRQCLEPCKKAGMRFGKCMN [1Nal5; 4952 GKCACTPK Ala34]OSK1WVIINVKCKISRQCLEPCKKAGMRFCKCMNGKCAC [Trp1; 4953 WPK Ala34]OSK1GVWINVKCKISRQCLEPCKKAGMRFGKCMNGKCAC [Trp3; 4954 TPK Ala34]OSK1GVIIWVKCKISRQCLEPCKKAGMRFGKCMNGKCAC [Trp5; 4955 TPK Ala34]OSK1WVWIWVKCKISRQCLEPCKKAGMRFGKCMNGKCAC [Trp1, 3, 5; 4956 TPK Ala34]OSK1[1Nal]V[1Nal]I[1Nal]VKCKISRQCLEPCKK [1Nal1, 3, 5; 4957AGMRFGKCMNGKCACTPK Ala34]OSK1 CKISRQCLEPCKKAGMRFGKCMNGKCACTPK Δ1-7, 4958[Ala34]OSK1 KCKISRQCLEPCKKAGMRFGKCMNGKCACTPK Δ1-6, 4959 [Ala34]OSK1GVIINVKCKI[1Nal]RQCLEPCKKAGMRFGKCAN [1Nal11; Ala29, 4960 GKCACWPK 34]Osk-1 GVIINVKCKIRRQCLEPCKKAGMRFGKCANGKCAC [Arg11; Ala29, 4961 WPK 34]Osk-1 GVIINVKCKISRQCEEPCKKAGMRFGKCANGKCAC [Glu15; Ala29, 4962 WPK 34]Osk-1 GVIINVKCKIRRQCLEPCKKAGMRFGKCMNGKCAC [Arg11; 4963 WPK Ala34]Osk-1GVIINVKCKISRQCEEPCKKAGMRFGKCMNGKCAC [Glu15; 4964 WPK Ala34]Osk-1CKIRRQCEEPCKKAGMRFGKCANGKCACTPK Δ1-7, [Arg11; 4965 Glu15; Ala29, 34]OSK1 GVIINVKCKIRRQCEEPCKKAGMRFGKCANGKCAC [Arg11; Glu15; 4966 TPKAla29, 34]OSK1 CVIINVKCKIRRQCEEPCKKAGMRFGKCANGKCAC [Cys1, 37; Arg11;4967 TCK Glu15; Ala29, 34]OSK1 GVIINVKCKIRAQCEEPCKKAGMRFGKCANGKCAC[Arg11; Ala12, 4968 TPK 29, 34; Glu15] OSK1GVIINVKCKIRAQCEEPCKKAGMRFGKCANGKCAC [Arg11; Glu15; 4969 TPK-NH2Ala12, 29, 34]OSK1- amide Ac- Ac-[Arg11; 4970GVIINVKCKIRAQCEEPCKKAGMRFGKCANGKCAC Glu15; Ala12, TPK-NH2 29, 34]OSK1-amide GVIINVKCKI[1Nal]AQCEEPCKKAGMRFGKCAN [1Nal11]; 4971 GKCACTPK Glu15;Ala12, 29, 34]OSK1 GVIINVKCKISRQCLEPCKKAGMRFGKCANGKC[1Nal][Ala29; 1Nal34] 4972 CWPK Osk-1 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHC[Ala12; Lys16; 4973 TPK Asp20]Osk-1 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[Ala12, 34; Lys16; 4974 TPK Asp20]Osk-1GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC [Ala12, 29, 34; Lys16; 4975 TPKAsp20]Osk-1 GVIINVKCKIRAQCLKPCKDAGMRFGKCANGKCAC [Arg11; Ala12, 4976 TPK29, 34; Lys16; Asp20] Osk-1 GVIINVKCKISAQCEKPCKDAGMRFGKCANGKCAC[Ala12, 29, 34; 4977 TPK Glu15, Lys16; Asp20]Osk-1GVIINVKCKI[1Nal]AQCLKPCKDAGMRFGKCAN [1Nal11; Ala12, 4978 GKCACTPK29, 34; Lys16; Asp20]Osk-1 GVIINVKCKIRAQCEKPCKDAGMRFGKCANGKCAC[Arg11; Ala12, 4979 TPK 29, 34; Glu15; Lys16; Asp20]Osk-1GVIINVKCKIRAQCEKPCKDAGMRFGKCMNGKCAC [Arg11; Ala12, 4980 TPK34; Glu15; Lys16; Asp20]Osk-1 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKC[1Nal][A12, K16, 4981 CTPK D20, Nal34]- OSK1GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC [A12, K16, 4982 TPK D20, A34]- OSK1GVIINVKCKISAQCLKPCKDAGMRFGKCANGKC[1Nal] [A12, K16, 4983 CTPK D20, A29,Nal34]-OSK1 GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC [A12, K16, 4984 TPKD20, A29, A34]-OSK1 {Acetyl}GVIINVKCKISRQCLEPCK(Glycyl) Ac- 4985KAGMRFGKCMNGKCACTPK [K(Gly)19, Ala34]- Osk1{Acetyl}GVIK(Glycyl)NVKCKISRQCLEPCK Ac- 4986 KAGMRFGKCMNGKCACTPK[K(Gly)4, Ala34]- Osk1 {Acetyl}GVIINVKCKISRQCLEPCKKAGMRFGK Ac- 4987CMNGKCACTPK(Glycyl) [Ala34, K(Gly) 38]-Osk1GVIINVKCKISRQCLEPCKKAGMRFGKCANGKC[1Nal] [A29, Nal34]- 4988 CTPK OSK1CKISRQCLKPCKDAGMRFGKCMNGKCHC{Amide} OSK1[des1-7, 4989 E16K,K20D, des36-38]- amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC OSK1- 4990TPK K16, D20, A34 GVIINVKCKI[1Nal]AQCLEPCKKAGMRFGKCAN Osk- 4991GKC[1Nal]CTPK 1[1Nal11, A12, A29, 1-Na134]GVIINVKCKI[1Nal]AQCLEPCKKAGMRFGKCAN [1Nal11, A12, A29, 4992GKC[1Nal]CTEK 1Nal34, E37] Osk-1 GVIINVKCKI[1Nal]AQCEEPCKKAGMRFGKCANOsk-1[1- 4993 GKC[1Nal]CEEK Nal11, A12, E15, A29, 1Nal34, E36, E37]GVIINVKCKI[1Nal]AQCLEPCKKAGFRFGKCAN [1Nal11, A12, F23, 4994GKC[1Nal]CTPK A29, 1Nal34] Osk-1 GVIINVKCKI[1Nal]AQCLEPCKKAG[Nle]RFG[1Nal11, A12, 4995 KCANGKC[1Nal]CTEK Nle23, A29, 1Nal34, E37]Osk-1GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCAC [Pro12, Lys16, 4996 TY[Nle] Asp20,Ala34, Tyr37, Nle38] Osk-1- amide GVIINVKCKISPQCLOPCKEAGMRFGKCMNGKCAC[P12, Orn16, E20, 4997 TY[Nle] A34, Y37, Nle38] Osk-1- amideNVKCKISRQCLEPCKKAGMRFGKCANGKC[1Nal] des1-4, [A29, 4998 CTPK Nal34]-OSK1NVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK des1-4, [A29, 4999 A34]-OSK1GVIINVKCKIRRQCLEPCKKAGMRFGKCANGKCAC [R11, A29, 5000 TPK A34]-OSK1GVIINVKCKIRAQCLEPCKKAGMRFGKCANGKCAC [R11, A12, 5001 TPK A29, A34]- OSK1CKISRQCLEPCKKAGMRFGKCMNGKCACTPK [Ala34]OSK1 5002 (8-35)CKISRQCLEPCKKAGMRFGKCMNGKCAC [Ala34]OSK1 5003 (8-35)CKIRRQCLEPCKKAGMRFGKCANGKCAC [Arg11; Ala29, 5004 34] Osk-1(8-35)CKISAQCLEPCKKAGMRFGKCANGKCAC [Ala12; Ala29, 5005 34] Osk-1(8-35)CKISAQCLEPCKKAGMRFGKCMNGKCAC [Ala12; Ala34] 5006 Osk-1(8-35)GVI[Dpr(AOA)]NVKCKISRQCLEPCKKAGMRFGKCM [Dpr^((AOA))4] 5009 NGKCHCTPKOsk1 GVI[Dpr ^((AOA-PEG)) ]NVKCKISRQCLEPCKKAGMRF [Dpr^((AOA)-PEG))4]5010 GKCMNGKCHCTPK Osk1 GCIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTCK[C2, C37]- 5012 OSK1 SCIINVKCKISRQCLEPCKKAGMRFGKCMNGRCHCTCK[S1, C2, C37]- 5013 OSK1 SCIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTCK[S1, C2, A34, 5014 C37]-OSK1 SCVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTCKSer-[C1, C37]- 5015 OSK1

Any OSK1 peptide analog that comprises an amino acid sequence selectedfrom SEQ ID NOS: 1391 through 4912, 4916, 4920 through 5006, 5009, 5010,and 5012 through 5015 as set forth in Tables 7, 7A, 7B, 7C, 7D, 7E, 7F,7G, 7H or 7I, is useful in accordance with the present invention. Any ofthese can also be derivatized at either its N-terminal or C-terminal,e.g., with a fatty acid having from 4 to 10 carbon atoms and from 0 to 2carbon-carbon double bonds, or a derivative thereof such as anω-amino-fatty acid. (E.g., Mouhat et al., WO 2006/002850 A2, which isincorporated by reference in its entirety). Examples of such fatty acidsinclude valeric acid or (for the C-terminal) co-amino-valeric acid.

Among useful OSK1 peptide analog sequences of the present invention areanalog sequences that introduce amino acid residues that can form anintramolecular covalent bridge (e.g., a disulfide bridge) ornon-covalent interactions (e.g. hydrophobic, ionic, stacking) betweenthe first and last beta strand, which may enhance the stability of thestructure of the unconjugated or conjugated (e.g., PEGylated) OSK1peptide analog molecule. Examples of such sequences include SEQ ID NOS:4985-4987 and 5012-5015.

In some embodiments of the composition of matter, the C-terminalcarboxylic acid moiety of the OSK1 peptide analog is replaced with amoiety selected from:

(A) —COOR, where R is independently (C₁-C₈)alkyl, haloalkyl, aryl orheteroaryl;

(B) —C(═O)NRR, where R is independently hydrogen, (C₁-C₈)alkyl,haloalkyl, aryl or heteroaryl; and

(C) —CH₂OR where R is hydrogen, (C₁-C₈) alkyl, aryl or heteroaryl.

“Aryl” is phenyl or phenyl vicinally-fused with a saturated,partially-saturated, or unsaturated 3-, 4-, or 5 membered carbon bridge,the phenyl or bridge being substituted by 0, 1, 2 or 3 substituentsselected from C₁₈ alkyl, C₁₄ haloalkyl or halo.

“Heteroaryl” is an unsaturated 5, 6 or 7 membered monocyclic orpartially-saturated or unsaturated 6-, 7-, 8-, 9-, 10- or 11 memberedbicyclic ring, wherein at least one ring is unsaturated, the monocyclicand the bicyclic rings containing 1, 2, 3 or 4 atoms selected from N, Oand S, wherein the ring is substituted by 0, 1, 2 or 3 substituentsselected from C₁₈ alkyl, C₁₄ haloalkyl and halo.

In other embodiments of the composition of matter comprising a half-lifeextending moiety, the OSK1 peptide analog comprises an amino acidsequence of the formula:

SEQ ID NO: 5011 G¹V²I³I⁴N⁵V⁶K⁷C⁸K⁹I¹⁰X_(aa) ¹¹X_(aa) ¹²Q¹³C¹⁴X_(aa)¹⁵X_(aa) ¹⁶P¹⁷ C¹⁸X_(aa) ¹⁹X_(aa) ²⁰A²¹G²²M²³R²⁴F²⁵G²⁶X_(aa) ²⁷C²⁸X_(aa)²⁹X_(aa) ³⁰ G³¹X_(aa) ³²C³³X_(aa) ³⁴C³⁵X_(aa) ³⁶X_(aa) ³⁷X_(aa) ³⁸

wherein:

amino acid residues 1 through 7 are optional (Thus, the OSK1 peptideanalog optionally includes residues 1-7 as indicated above in SEQ IDNO:5011, or a N-terminal truncation leaving present residues 2-7, 3-7,4-7, 5-7, 6-7, or 7, or alternatively, a N-terminal truncation whereinall of residues 1-7 are entirely absent.);

-   -   X_(aa) ¹¹ is a neutral, basic, or acidic amino acid residue        (e.g., Ser, Thr, Ala, Gly, Leu, Ile, Val, Met, Cit,        Homocitrulline, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Guf, and        4-Amino-Phe, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Lys, His, Trp,        Arg, N^(α) Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn,        Asn, Gln, Glu, Asp, α-aminoadipic acid, and        para-carboxyl-phenylalanine);    -   X_(aa) ¹² is a neutral or acidic amino acid residue (e.g., Ala,        Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz,        Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp,        α-aminoadipic acid, and para-carboxyl-phenylalanine);    -   X_(aa) ¹⁵ is a neutral or acidic amino acid residue (e.g., Ala,        Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz,        Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp,        α-aminoadipic acid, and para-carboxyl-phenylalanine);    -   X_(aa) ¹⁶ is a neutral or basic amino acid residue (e.g., Lys,        His, Arg, Trp, Arg, N^(α) Methyl-Arg; homoarginine, 1-Nal,        2-Nal, Orn, D-Orn, Cit, N^(α)-Methyl-Cit, Homocitrulline, His,        Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro,        Thz, Aib, Sar, Pip, Bip, Phe, Ser, Thr, Guf, and 4-Amino-Phe);    -   X_(aa) ¹⁹ is a neutral or basic amino acid residue (e.g., Lys,        His, Arg, Trp, Arg, N^(α) Methyl-Arg; homoarginine, 1-Nal,        2-Nal, Orn, D-Orn, Cit, N^(α)-Methyl-Cit, Homocitrulline, His,        Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro,        Thz, Aib, Sar, Pip, Bip, Phe, Ser, Thr, Guf, and 4-Amino-Phe);    -   X_(aa) ²⁰ is a neutral or basic amino acid residue (e.g., Lys,        His, Arg, Trp, Arg, N^(α) Methyl-Arg; homoarginine, 1-Nal,        2-Nal, Orn, D-Orn, Cit, N^(α)-Methyl-Cit, Homocitruiline, His,        Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro,        Thz, Aib, Sar, Pip, Bip, Phe, Ser, Thr, Guf, and 4-Amino-Phe);    -   X_(aa) ²⁷ is a neutral, basic, or acidic amino acid residue        (e.g., Ser, Thr, Ala, Gly, Leu, Ile, Val, Met, Cit,        Homocitrulline, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Guf, and        4-Amino-Phe, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Lys, His, Trp,        Arg, N^(α) Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn,        Asn, Gln, Glu, Asp, α-aminoadipic acid, and        para-carboxyl-phenylalanine);    -   X_(aa) ²⁹ is a neutral or acidic amino acid residue (e.g., Ala,        Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz,        Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp,        α-aminoadipic acid, and para-carboxyl-phenylalanine);    -   X_(aa) ³⁰ is a neutral or acidic amino acid residue (e.g., Ala,        Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz,        Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp,        α-aminoadipic acid, and para-carboxyl-phenylalanine);    -   X_(aa) ³² is a neutral, basic, or acidic amino acid residue        (e.g., Ser, Thr, Ala, Gly, Leu, Ile, Val, Met, Cit,        Homocitrulline, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Guf, and        4-Amino-Phe, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Lys, His, Trp,        Arg, N^(α) Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn,        Asn, Gln, Glu, Asp, α-aminoadipic acid, and        para-carboxyl-phenylalanine);    -   X_(aa) ³⁴ is a neutral or acidic amino acid residue (e.g., Ala,        Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz,        Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp,        α-aminoadipic acid, and para-carboxyl-phenylalanine);    -   X_(aa) ³⁶ is optional, and if present, is a neutral amino acid        residue (e.g., Pro, Ala, Gly, Leu, Ile, Val, Met, Oic, Hyp, Tic,        D-Tic, D-Pro, Thz, N^(α)-Methyl-Cit, Homocitrulline, Aib, Sar,        Pip, Tyr, Thr, Ser, Phe, Trp, 1-Nal, 2-Nal, and Bip;    -   X_(aa) ³⁷ is optional, and if present, is a neutral amino acid        residue (e.g., Pro, Ala, Gly, Leu, Ile, Val, Met, Oic, Hyp, Tic,        D-Tic, D-Pro, Thz, N^(α)-Methyl-Cit, Homocitrulline, Aib, Sar,        Pip, Tyr, Thr, Ser, Phe, Trp, 1-Nal, 2-Nal, and Bip); and    -   X_(aa) ³⁸ is optional, and if present, is a basic amino acid        residue (e.g., Lys, His, Orn, D-Orn, Arg, N^(α) Methyl-Arg;        homoarginine, Cit, N^(α)-Methyl-Cit, Homocitrulline, Guf, and        4-Amino-Phe).

In some other embodiments of the composition of matter comprising ahalf-life extending moiety, the OSK1 peptide analog comprises an aminoacid sequence of the formula:

SEQ ID NO: 4913 G¹V²I³I⁴N⁵V⁶K⁷C⁸K⁹I¹⁰X_(aa) ¹¹X_(aa) ¹²Q¹³C¹⁴L¹⁵X_(aa)¹⁶P¹⁷ C¹⁸K¹⁹X_(aa) ²⁰A²¹G²²M²³R²⁴F²⁵G²⁶X_(aa) ²⁷C^(28X) _(aa) ²⁹X_(aa)³⁰G³¹ K³²C³³X_(aa) ³⁴C³⁵X_(aa) ³⁶X_(aa) ³⁷X_(aa) ³⁸

wherein:

-   -   amino acid residues 1 to 7 are optional (Thus, the OSK1 peptide        analog optionally includes residues 1-7 as indicated above in        SEQ ID NO:4913, or a N-terminal truncation leaving present        residues 2-7, 3-7, 4-7, 5-7, 6-7, or 7, or alternatively, a        N-terminal truncation wherein all of residues 1-7 are entirely        absent.);    -   X_(aa) ¹¹ is a neutral, basic or acidic amino acid residue        (e.g., Ser, Thr, Ala, Gly, Leu, Ile, Val, Met, Cit,        Homocitrulline, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Guf, and        4-Amino-Phe, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Lys, His, Trp,        Arg, N^(α) Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn,        Asn, Gln, Glu, Asp, α-aminoadipic acid, and        para-carboxyl-phenylalanine);    -   X_(aa) ¹² is a neutral or acidic amino acid residue (e.g., Ala,        Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz,        Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp,        α-aminoadipic acid, and para-carboxyl-phenylalanine);    -   X_(aa) ¹⁶ is a neutral or basic amino acid residue (e.g., Lys,        His, Arg, Trp, Arg, N^(α) Methyl-Arg; homoarginine, 1-Nal,        2-Nal, Orn, D-Orn, Cit, N^(α)-Methyl-Cit, Homocitrulline, His,        Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro,        Thz, Aib, Sar, Pip, Bip, Phe, Ser, Thr, Guf, and 4-Amino-Phe);    -   X_(aa) ²⁰ is a neutral or basic amino acid residue (e.g., Lys,        His, Arg, Trp, Arg, N^(α) Methyl-Arg; homoarginine, 1-Nal,        2-Nal, Orn, D-Orn, Cit, N^(α)-Methyl-Cit, Homocitrulline, His,        Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro,        Thz, Aib, Sar, Pip, Bip, Phe, Ser, Thr, Guf, and 4-Amino-Phe);    -   X_(aa) ²⁷ is a neutral, basic, or acidic amino acid residue        (e.g., Ser, Thr, Ala, Gly, Leu, Ile, Val, Met, Cit,        Homocitrulline, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Guf, and        4-Amino-Phe, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Lys, His, Trp,        Arg, N^(α) Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn,        Asn, Gln, Glu, Asp, α-aminoadipic acid, and        para-carboxyl-phenylalanine);    -   X_(aa) ²⁹ is a neutral or acidic amino acid residue (e.g., Ala,        Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz,        Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp,        α-aminoadipic acid, and para-carboxyl-phenylalanine);    -   X_(aa) ³⁰ is a neutral or acidic amino acid residue (e.g., Ala,        Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz,        Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp,        α-aminoadipic acid, and para-carboxyl-phenylalanine);    -   X_(aa) ³⁵ is a neutral or acidic amino acid residue (e.g., Ala,        Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz,        Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp,        α-aminoadipic acid, and para-carboxyl-phenylalanine);    -   X_(aa) ³⁶ is optional, and if present, is a neutral amino acid        residue (e.g., Pro, Ala, Gly, Leu, Ile, Val, Met, Oic, Hyp, Tic,        D-Tic, D-Pro, Thz, N^(α)-Methyl-Cit, Homocitrulline, Aib, Sar,        Pip, Tyr, Thr, Ser, Phe, Trp, 1-Nal, 2-Nal, and Bip;    -   X_(aa) ³⁷ is optional, and if present, is a neutral amino acid        residue (e.g., Pro, Ala, Gly, Leu, Ile, Val, Met, Oic, Hyp, Tic,        D-Tic, D-Pro, Thz, N^(α)-Methyl-Cit, Homocitrulline, Aib, Sar,        Pip, Tyr, Thr, Ser, Phe, Trp, 1-Nal, 2-Nal, and Bip); and    -   X_(aa) ³⁸ is optional, and if present, is a basic amino acid        residue (e.g., Lys, His, Orn, D-Orn, Arg, N^(α) Methyl-Arg;        homoarginine, Cit, N^(α)-Methyl-Cit, Homocitrulline, Guf, and        4-Amino-Phe).

TABLE 8 Pi2 peptide and PiP2 s peptide analog equences Short- handdesig- SEQ ID Sequence/structure nation NO:TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFGR Pi2 17TISCTNPXQCYPHCKKETGYPNAKCMNRKCKCFGR Pi2-X8 299TISCTNPAQCYPHCKKETGYPNAKCMNRKCKCFGR Pi2-A8 300TISCTNPKQCYPHCXKETGYPNAKCMNRKCKCFGR Pi2-X15 301TISCTNPKQCYPHCAKETGYPNAKCMNRKCKCFGR Pi2-A15 302TISCTNPKQCYPHCKXETGYPNAKCMNRKCKCFGR Pi2-X16 303TISCTNPKQCYPHCKAETGYPNAKCMNRKCKCFGR Pi2-A16 304TISCTNPKQCYPHCKKETGYPNAXCMNRKCKCFGR Pi2-X24 305TISCTNPKQCYPHCKKETGYPNAACMNRKCKCFGR Pi2-A24 306TISCTNPKQCYPHCKKETGYPNAKCMNXKCKCFGR Pi2-X28 307TISCTNPKQCYPHCKKETGYPNAKCMNAKCKCFGR Pi2-A28 308TISCTNPKQCYPHCKKETGYPNAKCMNRXCKCFGR Pi2-X29 309TISCTNPKQCYPHCKKETGYPNAKCMNRACKCFGR Pi2-A29 310TISCTNPKQCYPHCKKETGYPNAKCMNRKCXCFGR Pi2-X31 311TISCTNPKQCYPHCKKETGYPNAKCMNRKCACFGR Pi2-A31 312TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFGX Pi2-X35 313TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFGA Pi2-A35 314TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFG Pi2-d35 315

TABLE 9 Anuroctoxin (AnTx) peptide and peptide analog sequences SEQShort-hand ID Sequence/structure designation NO:ZKECTGPQHCTNFCRKNKCTHGKCMNRKC Anuroctoxin 62 KCFNCK (AnTx)KECTGPQHCTNFCRKNKCTHGKCMNRKCKCFNCK AnTx-d1 316XECTGPQHCTNFCRKNKCTHGKCMNRKCKCFNCK AnTx-d1, X2 317AECTGPQHCTNFCRKNKCTHGKCMNRKCKCFNCK AnTx-d1, A2 318

TABLE 10 Noxiustoxin (NTX) peptide and NTX peptide analog sequencesShort-hand SEQ ID Sequence/structure designation NO:TIINVKCTSPKQCSKPCKELYGSSAGAKCMNG NTX 30 KCKCYNNTIINVACTSPKQCSKPCKELYGSSAGAKCMNG NTX-A6 319 KCKCYNNTIINVKCTSPKQCSKPCKELYGSSRGAKCMNG NTX-R25 320 KCKCYNNTIINVKCTSSKQCSKPCKELYGSSAGAKCMNG NTX-S10 321 KCKCYNNTIINVKCTSPKQCWKPCKELYGSSAGAKCMNG NTX-W14 322 KCKCYNNTIINVKCTSPKQCSKPCKELYGSSGAKCMNG NTX-A25d 323 KCKCYNNTIINVKCTSPKQCSKPCKELFGVDRGKCMNG NTX-IbTx1 324 KCKCYNNTIINVKCTSPKQCWKPCKELFGVDRGKCMNG NTX-IBTX2 325 KCKCYN

TABLE 11 Kaliotoxin1 (KTX1) peptide and KTX1 peptide analog sequencesShort-hand SEQ ID Sequence/structure designation NO:GVEINVKCSGSPQCLKPCKDAGMRFGKCMNRK KTX1 24 CHCTPK VRIPVSCKHSGQCLKPCKDAGMRFGKCMNGK KTX2 326 CDCTPKGVEINVSCSGSPQCLKPCKDAGMRFGKCMNRK KTX1-S7 327 CHCTPKGVEINVACSGSPQCLKPCKDAGMRFGKCMNRK KTX1-A7 328 CHCTPK

TABLE 12 IKCa1 inhibitor peptide sequences SEQ Short-hand IDSequence/structure designation NO: VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGCMTX 20   QFTNVSCTTSKECWSVCQRLHNTSRGKCMNKK ChTx 36 CRCYS  QFTQESCTASNQCWSICKRLHNTNRGKCMNKK ChTx-Lq2 329 CRCYS

TABLE 13 Maurotoxin (MTx) peptide amd MTx peptide analog sequencesShort- hand SEQ desig- ID Sequence/structure nation NO:VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX 20VSCAGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-A4 330VSCTGAKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-A6 331VSCTGSADCYAPCRKQTGCPNAKCINKSCKCYGC MTX-A7 332VSCTGSKDCAAPCRKQTGCPNAKCINKSCKCYGC MTX-A10 333VSCTGSKDCYAPCQKQTGCPNAKCINKSCKCYGC MTX-Q14 334VSCTGSKDCYAPCRQQTGCPNAKCINKSCKCYGC MTX-Q15 335VSCTGSKDCYAPCQQQTGCPNAKCINKSCKCYGC MTX-Q14, 336 15VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYAC MTX-A33 337VSCTGSKDCYAPCRKQTGCPYGKCMNRKCKCNRC MTX-HsTx1 338VSCTGSKDCYAACRKQTGCANAKCINKSCKCYGC MTX-A12, 339 20  VSCTGSKDCYAPCRKQTGX^(M19)PNAKCIN MTX-X19, 340 KSCKCYGX^(M34) 34VSCTGSKDCYAPCRKQTGSPNAKCINKSCKCYGS MTX-S19, 341 34VSCTGSADCYAPCRKQTGCPNAKCINKSCKCYGC MTX-A7 342VVIGQRCTGSKDCYAPCRKQTGCPNAKCINKSCK TsK-MTX 343 CYGCVSCRGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-R4 1301VSCGGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-G4 1302VSCTTSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-T5 1304VSCTASKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-A5 1305VSCTGTKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-T6 1306VSCTGPKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-P6 1307VSCTGSKDCGAPCRKQTGCPNAKCINKSCKCYGC MTX-G10 1309VSCTGSKDCYRPCRKQTGCPNAKCINKSCKCYGC MTX-R11 1311VSCTGSKDCYDPCRKQTGCPNAKCINKSCKCYGC MTX-D11 1312VSCTGSKDCYAPCRKRTGCPNAKCINKSCKCYGC MTX-R16 1315VSCTGSKDCYAPCRKETGCPNAKCINKSCKCYGC MTX-E16 1316VSCTGSKDCYAPCRKQTGCPYAKCINKSCKCYGC MTX-Y21 1317VSCTGSKDCYAPCRKQTGCPNSKCINKSCKCYGC MTX-S22 1318VSCTGSKDCYAPCRKQTGCPNGKCINKSCKCYGC MTX-G22 1319VSCTGSKDCYAPCRKQTGCPNAKCINRSCKCYGC MTX-R27 1320VSCTGSKDCYAPCRKQTGCPNAKCINKTCKCYGC MTX-T28 1321VSCTGSKDCYAPCRKQTGCPNAKCINKMCKCYGC MTX-M28 1322VSCTGSKDCYAPCRKQTGCPNAKCINKKCKCYGC MTX-K28 1323VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCNGC MTX-N32 1324VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYRC MTX-R33 1325VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-S35 1326 S SCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-d1 1327 SCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-S35 1328 S d1VSCTGSKDCYAPCAKQTGCPNAKCINKSCKCYGC MTX-A14 1329VSCTGSKDCYAPCRAQTGCPNAKCINKSCKCYGC MTX-A15 1330VSCTGSKDCYAPCRKQTGCPNAACINKSCKCYGC MTX-A23 1331VSCTGSKDCYAPCRKQTGCPNAKCINASCKCYGC MTX-A27 1332VSCTGSKDCYAPCRKQTGCPNAKCINKSCACYGC MTX-A30 1333VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCAGC MTX-A32 1334ASCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-A1 1335MSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-M1 1336

In Table 13 and throughout this specification, X^(m19) and X^(m34) areeach independently nonfunctional residues.

TABLE 14 Charybdotoxin(ChTx) peptide and ChTx peptide analog sequencesShort-hand Sequence/structure designation SEQ ID NO:QFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx 36QFTNVSCTTSKECWSVCQRLHNTSRGKCMNKECRCYS ChTx-E32 59QFTNVSCTTSKECWSVCQRLHNTSRGKCMNKDCRCYS ChTx-D32 344      CTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx-d1-d6 345QFTNVSCTTSKECWSVCQRLFGVDRGKCMGKKCRCYQ ChTx-IbTx 346QFTNVSCTTSKECWSVCQRLHNTSRGKCMNGKCRCYS ChTx-G31 1369QFTNVSCTTSKECLSVCQRLHNTSRGKCMNKKCRCYS ChTx-L14 1370QFTNVSCTTSKECASVCQRLHNTSRGKCMNKKCRCYS ChTx-A14 1371QFTNVSCTTSKECWAVCQRLHNTSRGKCMNKKCRCYS ChTx-A15 1372QFTNVSCTTSKECWPVCQRLHNTSRGKCMNKKCRCYS ChTx-P15 1373QFTNVSCTTSKECWSACQRLHNTSRGKCMNKKCRCYS ChTx-A16 1374QFTNVSCTTSKECWSPCQRLHNTSRGKCMNKKCRCYS ChTx-P16 1375QFTNVSCTTSKECWSVCQRLHNTSAGKCMNKKCRCYS ChTx-A25 1376QFTNVACTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx-A6 1377QFTNVKCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx-K6 1378QFTNVSCTTAKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx-A10 1379QFTNVSCTTPKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx-P10 1380QFTNVSCTTSKACWSVCQRLHNTSRGKCMNKKCRCYS ChTx-A12 1381QFTNVSCTTSKQCWSVCQRLHNTSRGKCMNKKCRCYS ChTx-Q12 1382AFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx-A1 1383TFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx-T1 1384QATNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx-A2 1385QITNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx-I2 1386QFANVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx-A3 1387QFINVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx-I3 1388TIINVKCTSPKQCLPPCKAQFGTSRGKCMNKKCRCYSP ChTx-MgTx 1389TIINVSCTSPKQCLPPCKAQFGTSRGKCMNKKCRCYSP ChTx-MgTx-b 1390

TABLE 15 SKCa inhibitor peptide sequences SEQ Short-hand IDSequence/structure designation NO:            CNCKAPETALCARRCQQHG Apamin68 AFCNLRMCQLSCRSLGLLGKCIGDKCECVKH ScyTx 51   AVCNLKRCQLSCRSLGLLGKCIGDKCECVKHG BmP05 50    TVCNLRRCQLSCRSLGLLGKCIGVKCECVKH P05 52    AFCNLRRCELSCRSLGLLGKCIGEECKCVPY Tamapin 53VSCEDCPEHCSTQKAQAKCDNDKCVCEPI P01 16 VVIGQRCYRSPDCYSACKKLVGKATGKCTNGRCDCTsK 47

TABLE 16 Apamin peptide and peptide analog inhibitor sequencesShort-hand SEQ Sequence/structure designation ID NO: CNCKAPETALCARRCQQHGApamin (Ap) 68 CNCXAPETALCARRCQQHG Ap-X4 348 CNCAAPETALCARRCQQHG Ap-A4349 CNCKAPETALCAXRCQQHG Ap-X13 350 CNCKAPETALCAARCQQHG Ap-A13 351CNCKAPETALCARXCQQHG Ap-X14 352 CNCKAPETALCARACQQHG Ap-A14 353

TABLE 17 Scyllatoxin (ScyTx), BmP05, P05, Tamapin, P01peptide and peptide analog inhibitor sequences Short-hand SEQ IDSequence/structure designation NO: AFCNLRMCQLSCRSLGLLGKCIGDKCECVKH ScyTx51 AFCNLRRCQLSCRSLGLLGKCIGDKCECVKH ScyTx-R7 354AFCNLRMCQLSCRSLGLLGKCMGKKCRCVKH ScyTx-IbTx 355AFSNLRMCQLSCRSLGLLGKSIGDKCECVKH ScyTx-C/S 356AFCNLRRCELSCRSLGLLGKCIGEECKCVPY Tamapin 53

TABLE 18 BKCa inhibitor peptide sequences Short-hand Sequence/structuredesignation SEQ ID NO: QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKKCRCYQ IbTx 38  TFIDVDCTVSKECWAPCKAAFGVDRGKCMGKKCKCYV Slotoxin 39 (SloTx)QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS BmTx1 40WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNKCRCYTN BuTx 41  FGLIDVKCFASSECWTACKKVTGSGQGKCQNNQCRCY MartenTx 35  ITINVKCTSPQQCLRPCKDRFGQHAGGKCINGKCKCYP CIITx1 29

TABLE 19 IbTx, Slotoxin, BmTx1, & BuTX (Slotoxin family) peptideand peptide analog inhibitor sequences Short-hand Sequence/structuredesignation SEQ ID NO: QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKKCRCYQ IbTx 38QFTDVDCSVSXECWSVCKDLFGVDRGKCMGKKCRCYQ IbTx-X11 357QFTDVDCSVSAECWSVCKDLFGVDRGKCMGKKCRCYQ IbTx-A11 358QFTDVDCSVSKECWSVCXDLFGVDRGKCMGKKCRCYQ IbTx-X18 359QFTDVDCSVSKECWSVCADLFGVDRGKCMCKKCRCYQ IbTx-A18 360QFTDVDCSVSKECWSVCKDLFGVDXGKCMGKKCRCYQ IbTx-X25 361QFTDVDCSVSKECWSVCKDLFGVDAGKCMGKKCRCYQ IbTx-A25 362QFTDVDCSVSKECWSVCKDLFGVDRGXCMGKKCRCYQ IbTx-X27 363QFTDVDCSVSKECWSVCKDLFGVDRGACMGKKCRCYQ IbTx-A27 364QFTDVDCSVSKECWSVCKDLFGVDRGKCMGXKCRCYQ IbTx-X31 365QFTDVDCSVSKECWSVCKDLFGVDRGKCMGAKCRCYQ IbTx-A31 366QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKXCRCYQ IbTx-X32 367QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKACRCYQ IbTx-A32 368QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKKCXCYQ IbTx-X34 369QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKKCACYQ IbTx-A34 370QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS BmTx1 371QFTDVXCTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS BmTx1-X6 372QFTDVACTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS BmTx1-A6 373QFTDVKCTGSXQCWPVCKQMFGKPNGKCMNGKCRCYS BmTx1-X11 374QFTDVKCTGSAQCWPVCKQMFGKPNGKCMNGKCRCYS BmTx1-A11 375QFTDVKCTGSKQCWPVCXQMFGKPNGKCMNGKCRCYS BmTx1-X18 376QFTDVKCTGSKQCWPVCAQMFGKPNGKCMNGKCRCYS BmTx1-A18 377QFTDVKCTGSKQCWPVCKQMFGXPNGKCMNGKCRCYS BmTx1-X23 378QFTDVKCTGSKQCWPVCKQMFGAPNGKCMNGKCRCYS BmTx1-A23 379QFTDVKCTGSKQCWPVCKQMFGKPNGXCMNGKCRCYS BmTx1-X27 380QFTDVKCTGSKQCWPVCKQMFGKPNGACMNGKCRCYS BmTx1-A27 381QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGXCRCYS BmTx1-X32 382QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGARCYS BmTx1-A32 383QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCXYS BmTx1-X34 384QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCAYS BmTx1-A34 385WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNKCRCYTN BuTx 386WCSTCLDLACGASXCYDPCFKAFGRAHGKCMNNKCRCYTN BuTx-X14 387WCSTCLDLACGASACYDPCFKAFGRAHGKCMNNKCRCYTN BuTx-A14 388WCSTCLDLACGASRECYDPCFXFGRAHGKCMNNKCRCYTN BuTx-X22 389WCSTCLDLACGASRECYDPCFAGRAHGKCMNNKCRCYTN BuTx-A22 390WCSTCLDLACGASRECYDPCFKAFGXHGKCMNNKCRCYTN BuTx-X26 391WCSTCLDLACGASRECYDPCFKAFGAHGKCMNNKCRCYTN BuTx-A26 392WCSTCLDLACGASRECYDPCFKAFGRAHGXMNNKCRCYTN BuTx-X30 393WCSTCLDLACGASRECYDPCFKAFGRAHGANNNKCRCYTN BuTx-A30 394WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNXRCYTN BuTx-X35 395WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNARCYTN BuTx-A35 396WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNKCXYTN BuTx-X37 397WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNKCAYTN BuTx-A37 398

TABLE 20 Martentoxin peptide and peptide analog inhibitor sequencesShort-hand Sequence/structure designation SEQ ID NO:FGLIDVKCFASSECWTACKKVTGSGQGKCQNNQCRCY MartenTx 35FGLIDVXCFASSECWTACKKVTGSGQGKCQNNQCRCY MartenTx-X7 399FGLIDVACFASSECWTACKKVTGSGQGKCQNNQCRCY MartenTx-A7 400FGLIDVKCFASSECWTACXKVTGSGQGKCQNNQCRCY MartenTx-X19 401FGLIDVKCFASSECWTACAKVTGSGQGKCQNNQCRCY MartenTx-A19 402FGLIDVKCFASSECWTACKXVTGSGQGKCQNNQCRCY MartenTx-X20 403FGLIDVKCFASSECWTACKAVTGSGQGKCQNNQCRCY MartenTx-A20 404FGLIDVKCFASSECWTACKKVTGSGQGXCQNNQCRCY MartenTx-X28 405FGLIDVKCFASSECWTACKKVTGSGQGACQNNQCRCY MartenTx-A28 406FGLIDVKCFASSECWTACKKVTGSGQGKCQNNQCXCY MartenTx-X35 407FGLIDVKCFASSECWTACKKVTGSGQGKCQNNQCACY MartenTx-A35 408

TABLE 21 N type Ca²⁺ channel inhibitor peptide sequences Short-hand des- SEQ ID Sequence/structure ignation NO:CKGKGAKCSRLMYDCCTGSCRSGKC MVIIA 65 CKSPGSSCSPTSYNCCRSCNPYTKRCY GVIA 64CKSKGAKCSKLMYDCCTGSCSGTVGRC CVIA 409 CKLKGQSCRKTSYDCCSGSCGRSGKC SVIB 347AEKDCIAPGAPCFGTDKPCCNPRAWCSSYANKCL Ptu1 66 CKGKGASCRKTMYDCCRGSCRSGRCCVIB 1364 CKGKGQSCSKLMYDCCTGSCSRRGKC CVIC 1365CKSKGAKCSKLMYDCCSGSCSGTVGRC CVID 1366 CLSXGSSCSXTSYNCCRSCNXYSRKCY TVIA1367

TABLE 22 ωMVIIA peptide and peptide analog inhibitor sequencesShort-hand Sequence/structure designation SEQ ID NO:CKGKGAKCSRLMYDCCTGSCRSGKC MVIIA 65 CXGKGAKCSRLMYDCCTGSCRSGKC MVIIA-X2410 CAGKGAKCSRLMYDCCTGSCRSGKC MVIIA-A2 411 CKGXGAKCSRLMYDCCTGSCRSGKCMVIIA-X4 412 CKGAGAKCSRLMYDCCTGSCRSGKC MVIIA-A4 413CKGKGAXCSRLMYDCCTGSCRSGKC MVIIA-X7 414 CKGKGAACSRLMYDCCTGSCRSGKCMVIIA-A7 415 CKGKGAKCSXLMYDCCTGSCRSGKC MVIIA-X10 416CKGKGAKCSALMYDCCTGSCRSGKC MVIIA-A10 417 CKGKGAKCSRLMYDCCTGSCXSGKCMVIIA-X21 418 CKGKGAKCSRLMYDCCTGSCASGKC MVIIA-A21 419CKGKGAKCSRLMYDCCTGSCRSGXC MVIIA-X24 420 CKGKGAKCSRLMYDCCTGSCRSGACMVIIA-A24 421

TABLE 23 □GVIA peptide and peptide analog inhibitor sequences Short-handSequence/structure designation SEQ ID NO: CKSPGSSCSPTSYNCCRSCNPYTKRCYGVIA 64 CXSPGSSCSPTSYNCCRSCNPYTKRCY GVIA-X2 422CASPGSSCSPTSYNCCRSCNPYTKRCY GVIA-A2 423 CKSPGSSCSPTSYNCCXSCNPYTKRCYGVIA-X17 424 CKSPGSSCSPTSYNCCASCNPYTKRCY GVIA-A17 425CKSPGSSCSPTSYNCCRSCNPYTXRCY GVIA-X24 426 CKSPGSSCSPTSYNCCRSCNPYTARCYGVIA-A24 427 CKSPGSSCSPTSYNCCRSCNPYTKXCY GVIA-X25 428CKSPGSSCSPTSYNCCRSCNPYTKACY GVIA-A25 429

TABLE 24 Ptu1 peptide and peptide analog inhibitor sequences Short-hand des- SEQ ID Sequence/structure ignation NO:AEKDCIAPGAPCFGTDKPCCNPRAWCSSYANKCL Ptu1 66AEXDCIAPGAPCFGTDKPCCNPRAWCSSYANKCL Ptu1-X3 430AEADCIAPGAPCFGTDKPCCNPRAWCSSYANKCL Ptu1-A3 431AEKDCIAPGAPCFGTDXPCCNPRAWCSSYANKCL Ptu1-X17 432AEKDCIAPGAPCFGTDAPCCNPRAWCSSYANKCL Ptu1-A17 433AEKDCIAPGAPCFGTDKPCCNPXAWCSSYANKCL Ptu1-X23 434AEKDCIAPGAPCFGTDKPCCNPAAWCSSYANKCL Ptu1-A23 435AEKDCIAPGAPCFGTDKPCCNPRAWCSSYANXCL Ptu1-X32 436AEKDCIAPGAPCFGTDKPCCNPRAWCSSYANACL Ptu1-A32 437

TABLE 25 Thrixopelma pruriens (ProTx1) and ProTx1 peptide analogsand other T type Ca²⁺ channel inhibitor peptide sequences Short-handSequence/structure designation SEQ ID NO:ECRYWLGGCSAGQTCCKHLVCSRRHGWCVWDGTFS ProTx1 56ECXYWLGGCSAGQTCCKHLVCSRRHGWCVWDGTFS ProTx1-X3 438ECAYWLGGCSAGQTCCKHLVCSRRHGWCVWDGTFS ProTx1-A3 439ECRYWLGGCSAGQTCCXHLVCSRRHGWCVWDGTFS ProTx1-X17 440ECRYWLGGCSAGQTCCAHLVCSRRHGWCVWDGTFS ProTx1-A17 441ECRYWLGGCSAGQTCCKHLVCSXRHGWCVWDGTFS ProTx1-X23 442ECRYWLGGCSAGQTCCKHLVCSARHGWCVWDGTFS ProTx1-A23 443ECRYWLGGCSAGQTCCKHLVCSRXHGWCVWDGTFS ProTx1-X24 444ECRYWLGGCSAGQTCCKHLVCSRAHGWCVWDGTFS ProTx1-A24 445KIDGYPVDYW NCKRICWYNN KYCNDLCKGL Kurtoxin 1276KADSGYCWGW TLSCYCQGLP DNARIKRSGR CRA

TABLE 26 BeKM1 M current inhibitor peptide and BeKM1peptide analog sequences Short-hand Sequence/structure designationSEQ ID NO: RPTDIKCSESYQCFPVCKSRFGKTNGRCVNGFCDCF BeKM1 63 PTDIKCSESYQCFPVCKSRFGKTNGRCVNGFCDCF BeKM1-d1 446XPTDIKCSESYQCFPVCKSRFGKTNGRCVNGFCDCF BeKM1-X1 447APTDIKCSESYQCFPVCKSRFGKTNGRCVNGFCDCF BeKM1-A1 448RPTDIXCSESYQCFPVCKSRFGKTNGRCVNGFCDCF BeKM1-X6 449RPTDIACSESYQCFPVCKSRFGKTNGRCVNGFCDCF BeKM1-A6 450RPTDIKCSESYQCFPVCXSRFGKTNGRCVNGFCDCF BeKM1-X18 451RPTDIKCSESYQCFPVCASRFGKTNGRCVNGFCDCF BeKM1-A18 452RPTDIKCSESYQCFPVCKSXFGKTNGRCVNGFCDCF BeKM1-X20 453RPTDIKCSESYQCFPVCKSAFGKTNGRCVNGFCDCF BeKM1-A20 454RPTDIKCSESYQCFPVCKSRFGXTNGRCVNGFCDCF BeKM1-X23 455RPTDIKCSESYQCFPVCKSRFGATNGRCVNGFCDCF BeKM1-A23 456RPTDIKCSESYQCFPVCKSRFGKTNGXCVNGFCDCF BeKM1-X27 457RPTDIKCSESYQCFPVCKSRFGKTNGACVNGFCDCF BeKM1-A27 458

TABLE 27 Na⁺ channel inhibitor peptide sequences Short-hand SEQ IDSequence/structure designation NO: QRCCNGRRGCSSRWCRDHSRCC SmIIIa 459RDCCTOOKKCKDRQCKOQRCCA μ-GIIIA 460 RDCCTOORKCKDRRCKOMRCCA μ-GIIIB 461ZRLCCGFOKSCRSRQCKOHRCC μ-PIIIA 462 ZRCCNGRRGCSSRWCRDHSRCC μ-SmIIIA 463ACRKKWEYCIVPIIGFIYCCPGLICGPFVCV μO-MrVIA 464ACSKKWEYCIVPIIGFIYCCPGLICGPFVCV μO-MrVIB 465EACYAOGTFCGIKOGLCCSEFCLPGVCFG δ-PVIA 466 DGCSSGGTFCGIHOGLCCSEFCFLWCITFIDδ-SVIE 467 WCKQSGEMCNLLDQNCCDGYCIVLVCT δ-TxVIA 468VKPCRKEGQLCDPIFQNCCRGWNCVLFCV δ-GmVIA 469

TABLE 28 Cl⁻ channel inhibitor peptide sequences Short-handSequence/structure designation SEQ ID NO:  MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR CTX 67MCMPCFTTDHQMAXKCDDCCGGKGRGKCYGPQCLCR CTX-X14 470MCMPCFTTDHQMAAKCDDCCGGKGRGKCYGPQCLCR CTX-A14 471MCMPCFTTDHQMARXCDDCCGGKGRGKCYGPQCLCR CTX-X15 472MCMPCFTTDHQMARACDDCCGGKGRGKCYGPQCLCR CTX-A15 473MCMPCFTTDHQMARKCDDCCGGXGRGKCYGPQCLCR CTX-X23 474MCMPCFTTDHQMARKCDDCCGGAGRGKCYGPQCLCR CTX-A23 475MCMPCFTTDHQMARKCDDCCGGKGXGKCYGPQCLCR CTX-X25 476MCMPCFTTDHQMARKCDDCCGGKGAGKCYGPQCLCR CTX-A25 477MCMPCFTTDHQMARKCDDCCGGKGRGXCYGPQCLCR CTX-X27 478MCMPCFTTDHQMARKCDDCCGGKGRGACYGPQCLCR CTX-A27 479  MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCX CTX-X36 480MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCA CTX-A36 481   MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLC CTX-d36 482QTDGCGPCFTTDANMARKCRECCGGNGKCFGPQCLCNRE Bm-12b 483QTDGCGPCFTTDANMAXKCRECCGGNGKCFGPQCLCNRE Bm-12b-X17 484QTDGCGPCFTTDANMAAKCRECCGGNGKCFGPQCLCNRE Bm-12b-A17 485QTDGCGPCFTTDANMARXCRECCGGNGKCFGPQCLCNRE Bm-12b-X18 486QTDGCGPCFTTDANMARACRECCGGNGKCFGPQCLCNRE Bm-12b-A18 487QTDGCGPCFTTDANMARKCXECCGGNGKCFGPQCLCNRE Bm-12b-X20 488 QTDGCGPCFTTDANMARKCAECCGGNGKCFGPQCLCNRE Bm-12b-A20 489QTDGCGPCFTTDANMARKCRECCGGNGXCFGPQCLCNRE Bm-12b-X28 490QTDGCGPCFTTDANMARKCRECCGGNGACFGPQCLCNRE Bm-12b-A28 491QTDGCGPCFTTDANMARKCRECCGGNGKCFGPQCLCNXE Bm-12b-X38 492QTDGCGPCFTTDANMARKCRECCGGNGKCFGPQCLCNAE Bm-12b-A38 493

TABLE 29 Kv2.1 inhibitor peptide sequences Short-hand Sequence/structuredesignation SEQ ID NO: ECRYLFGGCKTTSDCCKHLGCKFRDKYCAWDFTFS HaTx1 494ECXYLFGGCKTTSDCCKHLGCKFRDKYCAWDFTFS HaTx1-X3 495ECAYLFGGCKTTSDCCKHLGCKFRDKYCAWDFTFS HaTx1-A3 496ECRYLFGGCXTTSDCCKHLGCKFRDKYCAWDFTFS HaTx1-X10 497ECRYLFGGCATTSDCCKHLGCKFRDKYCAWDFTFS HaTx1-A10 498ECRYLFGGCKTTSDCCXHLGCKFRDKYCAWDFTFS HaTx1-X17 499ECRYLFGGCKTTSDCCAHLGCKFRDKYCAWDFTFS HaTx1-A17 500ECRYLFGGCKTTSDCCKHLGCXFRDKYCAWDFTFS HaTx1-X22 501ECRYLFGGCKTTSDCCKHLGCAFRDKYCAWDFTFS HaTx1-A22 502ECRYLFGGCKTTSDCCKHLGCKFXDKYCAWDFTFS HaTx1-X24 503ECRYLFGGCKTTSDCCKHLGCKFADKYCAWDFTFS HaTx1-A24 504ECRYLFGGCKTTSDCCKHLGCKFRDXYCAWDFTFS HaTx1-X26 505ECRYLFGGCKTTSDCCKHLGCKFRDAYCAWDFTFS HaTx1-A26 506

TABLE 30 Kv4.3 & Kv4.2 inhibitor peptide sequences Short-hand SEQ IDSequence/structure designation NO: YCQKWMWTCDEERKCCEGLVCRLWCKRIINM PaTx257 YCQXWMWTCDEERKCCEGLVCRLWCKRIINM PaTx2-X4 507YCQAWMWTCDEERKCCEGLVCRLWCKRIINM PaTx2-A4 508YCQKWMWTCDEEXKCCEGLVCRLWCKRIINM PaTx2-X13 509YCQKWMWTCDEEAKCCEGLVCRLWCKRIINM PaTx2-A13 510YCQKWMWTCDEERXCCEGLVCRLWCKRIINM PaTx2-X14 511YCQKWMWTCDEERACCEGLVCRLWCKRIINM PaTx2-A14 512YCQKWMWTCDEERKCCEGLVCXLWCKRIINM PaTx2-X22 513YCQKWMWTCDEERKCCEGLVCALWCKRIINM PaTx2-A22 514YCQKWMWTCDEERKCCEGLVCRLWCXRIINM PaTx2-X26 515YCQKWMWTCDEERKCCEGLVCRLWCARIINM PaTx2-A26 516YCQKWMWTCDEERKCCEGLVCRLWCKXIINM PaTx2-X27 517YCQKWMWTCDEERKCCEGLVCRLWCKAIINM PaTx2-A27 518

TABLE 31 nACHR channel inhibitor peptide sequences SEQ Short-hand IDSequence/structure designation NO: GCCSLPPCAANNPDYC PnIA 519GCCSLPPCALNNPDYC PnIA-L10 520 GCCSLPPCAASNPDYC PnIA-S11 521GCCSLPPCALSNPDYC PnIB 522 GCCSLPPCAASNPDYC PnIB-A10 523 GCCSLPPCALNNPDYCPnIB-N11 524 GCCSNPVCHLEHSNLC MII 525 GRCCHPACGKNYSC α-MI 526RD(hydroxypro)CCYHPTCNMSNPQIC α-EI 527 GCCSYPPCFATNPDC α-AuIB 528RDPCCSNPVCTVHNPQIC α-PIA 529 GCCSDPRCAWRC α-ImI 530 ACCSDRRCRWRC α-ImII531 ECCNPACGRHYSC α-GI 532 GCCGSY(hydroxypro)NAACH(hydroxypro) αA-PIVA533 CSCKDR(hydroxypro)SYCGQ GCCPY(hydroxypro) αA-EIVA 534NAACH(hydroxypro)CGCKVCR (hydroxypro)(hydroxypro) YCDR(hydroxypro)SGGH(hydroxypro)(hydroxypro) ψ-PIIIE 535 CCLYGKCRRY(hydroxypro) GCSSASCCQRGCCSDPRCNMNNPDYC EpI 536 GCCSHPACAGNNQHIC GIC 537IRD(γ-carboxyglu) CCSNPACRVNN GID 538 (hydroxypro)HVC GGCCSHPACAANNQDYCAnIB 539 GCCSYPPCFATNSDYC AuIA 540 GCCSYPPCFATNSGYC AuIC 541

TABLE 32 Agelenopsis aperta (Agatoxin) toxin peptides and peptideanalogs and other Ca²⁺ channel inhibiter peptides Short-handSequence/structure designation SEQ ID NO:KKKCIAKDYG RCKWGGTPCC RGRGCICSIM ω-Aga-IVA 959 GTNCECKPRL IMEGLGLAEDNCIAEDYG KCTWGGTKCC RGRPCRCSMI ω-Aga-IVB 960 GTNCECTPRL IMEGLSFASCIDIGGDCD GEKDDCQCCR RNGYCSCYSL ω-Aga-IIIA 961FGYLKSGCKC VVGTSAEFQG ICRRKARQCY NSDPDKCESH NKPKRRSCIDIGGDCD GEKDDCQCCR RNGYCSCYSL ω-Aga-IIIA- 962FGYLKSGCKC VVGTSAEFQG ICRRKARTCY T58 NSDPDKCESH NKPKRRSCIDFGGDCD GEKDDCQCCR SNGYCSCYSL ω-Aga-IIIB 963FGYLKSGCKC EVGTSAEFRR ICRRKAKQCY NSDPDKCVSV YKPKRRSCIDFGGDCD GEKDDCQCCR SNGYCSCYNL ω-Aga-IIIB- 964 FGYLKSGCKC EVGTSAEFRRN29 ICRRKAKQCYNSDPDKCVSV YKPKRR SCIDFGGDCD GEKDDCQCCR SNGYCSCYNLω-Aga-IIIB- 965 FGYLRSGCKC EVGTSAEFRR ICRRKAKQCY N29/R35NSDPDKCVSV YKPKRR NCIDFGGDCD GEKDDCQCCX RNGYCSCYNL ω-Aga-IIIC 966FGYLKRGCKX EVG SCIKIGEDCD GDKDDCQCCR TNGYCSXYXL FGYLKSG ω-Aga-IIID 967GCIEIGGDCD GYQEKSYCQC CRNNGFCS ω-Aga-IIA 968AKAL PPGSVCDGNE SDCKCYGKWH KCRCPWKWHF ω-Aga-IA 969TGEGPCTCEK GMKHTCITKL HCPNKAEWGL DW (major chain)ECVPENGHCR DWYDECCEGF YCSCRQPPKC ICRNNNX μ-Aga 970DCVGESQQCA DWAGPHCCDG YYCTCRYFPK CICVNNN μ-Aga-6 971ACVGENKQCA DWAGPHCCDG YYCTCRYFPK CICRNNN μ-Aga-5 972ACVGENQQCA DWAGPHCCDG YYCTCRYFPK CICRNNN μ-Aga-4 973ADCVGDGQRC ADWAGPYCCS GYYCSCRSMP μ-Aga-3 1275 YCRCRSDSECATKNKRCA DWAGPWCCDG LYCSCRSYPG CMCRPSS μ-Aga-2 974ECVPENGHCR DWYDECCEGF YCSCRQPPKC ICRNNN μ-Aga-1 975AELTSCFPVGHECDGDASNCNCCGDDVYCGCGWGRWNCKC Tx-1 1277KVADQSYAYGICKDKVNCPNRHLWPAKVCKKPCRREC GCANAYKSCNGPHTCCWGYNGYKKACICSGXNWKTx3-3 1278 SCINVGDFCDGKKDCCQCDRDNAFCSCSVIFGYKTNCRCE Tx3-4 1279SCINVGDFCDGKKDDCQCCRDNAFCSCSVIFGYKTNCRCE ω-PtXIIA 1280VGTTATSYGICMAKHKCGRQTTCTKPCLSKRCKKNHAECLMIGDTSCVPRLGRRCCYGAWCYCDQQLSCRRVGRKR Dw13.3 1281ECGWVEVNCKCGWSWSQRIDDWRADYSCKCPEDQ GGCLPHNRFCNALSGPRCCSGLKCKELSIWDSRCLAgelenin 1282 DCVRFWGKCSQTSDCCPHLACKSKWPRNICVWDGSV ω-GTx-SIA 1283GCLEVDYFCG IPFANNGLCC SGNCVFVCTP Q ω-conotoxin 1284 PnVIADDDCEPPGNF CGMIKIGPPC CSGWCFFACA ω-conotoxin 1285 PnVIB VCCGYKLCHP CLambda- 1286 conotoxin CMrVIA MRCLPVLIIL LLLTASAPGV VVLPKTEDDV Lambda-1287 PMSSVYGNGK SILRGILRNG VCCGYKLCHP C conotoxin CMrVIBKIDGYPVDYW NCKRICWYNN KYCNDLCKGL Kurtoxin 1276KADSGYCWGW TLSCYCQGLP DNARIKRSGR CRA CKGKGAPCRKTMYDCCSGSCGRRGKC MVIIC1368

In accordance with this invention are molecules in which at least one ofthe toxin peptide (P) portions of the molecule comprises a Kv1.3antagonist peptide. Amino acid sequences selected from ShK, HmK, MgTx,AgTx1, AgTx2, Heterometrus spinnifer (HsTx1), OSK1, Anuroctoxin (AnTx),Noxiustoxin (NTX), KTX1, Hongotoxin, ChTx, Titystoxin, BgK, BmKTX, BmTx,AeK, AsKS Tc30, Tc32, Pi1, Pi2, and/or Pi3 toxin peptides and peptideanalogs of any of these are preferred. Examples of useful Kv1.3antagonist peptide sequences include those having any amino acidsequence set forth in Table 1, Table 2, Table 3, Table 4, Table 5, Table6, Table 7, Table 8, Table 9, Table 10, and/or Table 11 herein above;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is an IKCa1 antagonist peptide. Useful IKCa1antagonist peptides include Maurotoxin (MTx), ChTx, peptides and peptideanalogs of either of these, examples of which include those having anyamino acid sequence set forth in Table 12, Table 13, and/or Table 14;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a SKCa inhibitor peptide. Useful SKCainhibitor peptides include, Apamin, ScyTx, BmP05, P01, P05, Tamapin,TsK, and peptide analogs of any of these, examples of which includethose having any amino acid sequence set forth in Table 15;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is an apamin peptide, and peptide analogs ofapamin, examples of which include those having any amino acid sequenceset forth in Table 16;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a Scyllotoxin family peptide, and peptideanalogs of any of these, examples of which include those having anyamino acid sequence set forth in Table 17;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a BKCa inhibitor peptide, examples of whichinclude those having any amino acid sequence set forth in Table 18;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a Slotoxin family peptide, and peptide analogsof any of these, examples of which include those having any amino acidsequence set forth in Table 19;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a Martentoxin peptide, and peptide analogsthereof, examples of which include those having any amino acid sequenceset forth in Table 20;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a N-type Ca²⁺ channel inhibitor peptide,examples of which include those having any amino acid sequence set forthin Table 21;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a ωMVIIA peptide, and peptide analogs thereof,examples of which include those having any amino acid sequence set forthin Table 22;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a ωGVIA peptide, and peptide analogs thereof,examples of which include those having any amino acid sequence set forthin Table 23;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a Ptu1 peptide, and peptide analogs thereof,examples of which include those having any amino acid sequence set forthin Table 24;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a ProTx1 peptide, and peptide analogs thereof,examples of which include those having any amino acid sequence set forthin Table 25;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a BeKM1 peptide, and peptide analogs thereof,examples of which include those having any amino acid sequence set forthin Table 26;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a Na⁺ channel inhibitor peptide, examples ofwhich include those having any amino acid sequence set forth in Table27;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a Cl⁻ channel inhibitor peptide, examples ofwhich include those having any amino acid sequence set forth in Table28;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a Kv2.1 inhibitor peptide, examples of whichinclude those having any amino acid sequence set forth in Table 29;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a Kv4.2/Kv4.3 inhibitor peptide, examples ofwhich include those having any amino acid sequence set forth in Table30;

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is a nACHR inhibitor peptide, examples of whichinclude those having any amino acid sequence set forth in Table 31; and

Other embodiments of the inventive composition include at least onetoxin peptide (P) that is an Agatoxin peptide, a peptide analog thereofor other calcium channel inhibitor peptide, examples of which includethose having any amino acid sequence set forth in Table 32.

Half-life extending moieties. This invention involves the presence of atleast one half-life extending moiety (F¹ and/or F² in Formula I)attached to a peptide through the N-terminus, C-terminus or a sidechainof one of the intracalary amino acid residues. Multiple half-lifeextending moieties can also be used; e.g., Fc's at each terminus or anFc at a terminus and a PEG group at the other terminus or at asidechain. In other embodiments the Fc domain can be PEGylated (e.g., inaccordance with the formulae F¹-F²-(L)_(f)-P; P-(L)_(g)-F¹-F²; orP-(L)₉-F¹-F²-(L)_(f)-P).

The half-life extending moiety can be selected such that the inventivecomposition achieves a sufficient hydrodynamic size to prevent clearanceby renal filtration in vivo. For example, a half-life extending moietycan be selected that is a polymeric macromolecule, which issubstantially straight chain, branched-chain, or dendritic in form.Alternatively, a half-life extending moiety can be selected such that,in vivo, the inventive composition of matter will bind to a serumprotein to form a complex, such that the complex thus formed avoidssubstantial renal clearance. The half-life extending moiety can be, forexample, a lipid; a cholesterol group (such as a steroid); acarbohydrate or oligosaccharide; or any natural or synthetic protein,polypeptide or peptide that binds to a salvage receptor.

Exemplary half-life extending moieties that can be used, in accordancewith the present invention, include an immunoglobulin Fc domain, or aportion thereof, or a biologically suitable polymer or copolymer, forexample, a polyalkylene glycol compound, such as a polyethylene glycolor a polypropylene glycol. Other appropriate polyalkylene glycolcompounds include, but are not limited to, charged or neutral polymersof the following types: dextran, polylysine, colominic acids or othercarbohydrate based polymers, polymers of amino acids, and biotinderivatives. In some monomeric fusion protein embodiments animmunoglobulin (including light and heavy chains) or a portion thereof,can be used as a half-life-extending moiety, preferably animmunoglobulin of human origin, and including any of theimmunoglobulins, such as, but not limited to, IgG1, IgG2, IgG3 or IgG4.

Other examples of the half-life extending moiety, in accordance with theinvention, include a copolymer of ethylene glycol, a copolymer ofpropylene glycol, a carboxymethylcellulose, a polyvinyl pyrrolidone, apoly-1,3-dioxolane, a poly-1,3,6-trioxane, an ethylene/maleic anhydridecopolymer, a polyaminoacid (e.g., polylysine), a dextran n-vinylpyrrolidone, a poly n-vinyl pyrrolidone, a propylene glycol homopolymer,a propylene oxide polymer, an ethylene oxide polymer, a polyoxyethylatedpolyol, a polyvinyl alcohol, a linear or branched glycosylated chain, apolyacetal, a long chain fatty acid, a long chain hydrophobic aliphaticgroup, an immunoglobulin light chain and heavy chain, an immunoglobulinFc domain or a portion thereof (see, e.g., Feige et al., Modifiedpeptides as therapeutic agents, U.S. Pat. No. 6,660,843), a CH2 domainof Fc, an albumin (e.g., human serum albumin (HSA)); see, e.g., Rosen etal., Albumin fusion proteins, U.S. Pat. No. 6,926,898 and US2005/0054051; Bridon et al., Protection of endogenous therapeuticpeptides from peptidase activity through conjugation to bloodcomponents, U.S. Pat. No. 6,887,470), a transthyretin (TTR; see, e.g.,Walker et al., Use of transthyretin peptide/protein fusions to increasethe serum half-life of pharmacologically active peptides/proteins, US2003/0195154 A1; 2003/0191056 A1), or a thyroxine-binding globulin(TBG). Thus, exemplary embodiments of the inventive compositions alsoinclude HSA fusion constructs such as but not limited to: HSA fusionswith ShK, OSK1, or modified analogs of those toxin peptides. Examplesinclude HSA-L10-ShK(2-35); HSA-L10-OsK1(1-38); HSA-L10-ShK(2-35); andHSA-L10-OsK1(1-38).

Other embodiments of the half-life extending moiety, in accordance withthe invention, include peptide ligands or small (organic) moleculeligands that have binding affinity for a long half-life serum proteinunder physiological conditions of temperature, pH, and ionic strength.Examples include an albumin-binding peptide or small molecule ligand, atransthyretin-binding peptide or small molecule ligand, athyroxine-binding globulin-binding peptide or small molecule ligand, anantibody-binding peptide or small molecule ligand, or another peptide orsmall molecule that has an affinity for a long half-life serum protein.(See, e.g., Blaney et al., Method and compositions for increasing theserum half-life of pharmacologically active agents by binding totransthyretin-selective ligands, U.S. Pat. No. 5,714,142; Sato et al.,Serum albumin binding moieties, US 2003/0069395 A1; Jones et al.,Pharmaceutical active conjugates, U.S. Pat. No. 6,342,225). A “longhalf-life serum protein” is one of the hundreds of different proteinsdissolved in mammalian blood plasma, including so-called “carrierproteins” (such as albumin, transferrin and haptoglobin), fibrinogen andother blood coagulation factors, complement components, immunoglobulins,enzyme inhibitors, precursors of substances such as angiotensin andbradykinin and many other types of proteins. The invention encompassesthe use of any single species of pharmaceutically acceptable half-lifeextending moiety, such as, but not limited to, those described herein,or the use of a combination of two or more different half-life extendingmoieties, such as PEG and immunoglobulin Fc domain or a CH2 domain ofFc, albumin (e.g., HSA), an albumin-binding protein, transthyretin orTBG, or a combination such as immunoglobulin (light chain+heavy chain)and Fc domain (the combination so-called “hemibody”).

In some embodiments of the invention an Fc domain or portion thereof,such as a CH2 domain of Fc, is used as a half-life extending moiety. TheFc domain can be fused to the N-terminal (e.g., in accordance with theformula F¹-(L)_(f)-P) or C-terminal (e.g., in accordance with theformula P-(L)_(g)-F¹) of the toxin peptides or at both the N and Ctermini (e.g., in accordance with the formulae F¹-(L)_(f)-P-(L)_(g)-F²or P-(L)_(g)-F¹-(L)_(f)-P). A peptide linker sequence can be optionallyincluded between the Fc domain and the toxin peptide, as describedherein. Examples of the formula F¹-(L)_(f)-P include:Fc-L10-ShK(K22A)[2-35]; Fc-L10-ShK(R1K/K22A)[1-35];Fc-L10-ShK(R1H/K22A)[1-35]; Fc-L110-ShK(R1Q/K22A)[1-35];Fc-L110-ShK(R1Y/K22A)[1-35]; Fc-L10-PP-ShK(K22A) [1-35]; and any otherworking examples described herein. Examples of the formula P-(L)_(g)-F¹include: ShK(1-35)-L10-Fc; OsK1(1-38)-L10-Fc; Met-ShK(1-35)-L10-Fc;ShK(2-35)-L10-Fc; Gly-ShK(1-35)-L10-Fc; Osk1(1-38)-L10-Fc; and any otherworking examples described herein.

Fc variants are suitable half-life extending moieties within the scopeof this invention. A native Fc can be extensively modified to form an Fcvariant in accordance with this invention, provided binding to thesalvage receptor is maintained; see, for example WO 97/34631, WO96/32478, and WO 04/110 472. In such Fc variants, one can remove one ormore sites of a native Fc that provide structural features or functionalactivity not required by the fusion molecules of this invention. One canremove these sites by, for example, substituting or deleting residues,inserting residues into the site, or truncating portions containing thesite. The inserted or substituted residues can also be altered aminoacids, such as peptidomimetics or D-amino acids. Fc variants can bedesirable for a number of reasons, several of which are described below.Exemplary Fc variants include molecules and sequences in which:

-   -   1. Sites involved in disulfide bond formation are removed. Such        removal can avoid reaction with other cysteine-containing        proteins present in the host cell used to produce the molecules        of the invention. For this purpose, the cysteine-containing        segment at the N-terminus can be truncated or cysteine residues        can be deleted or substituted with other amino acids (e.g.,        alanyl, seryl). In particular, one can truncate the N-terminal        20-amino acid segment of SEQ ID NO: 2 or delete or substitute        the cysteine residues at positions 7 and 10 of SEQ ID NO: 2.        Even when cysteine residues are removed, the single chain Fc        domains can still form a dimeric Fc domain that is held together        non-covalently.    -   2. A native Fc is modified to make it more compatible with a        selected host cell. For example, one can remove the PA sequence        near the N-terminus of a typical native Fc, which can be        recognized by a digestive enzyme in E. coli such as proline        iminopeptidase. One can also add an N-terminal methionine        residue, especially when the molecule is expressed recombinantly        in a bacterial cell such as E. coli. The Fc domain of SEQ ID NO:        2 (FIG. 4A-4B) is one such Fc variant.    -   3. A portion of the N-terminus of a native Fc is removed to        prevent N-terminal heterogeneity when expressed in a selected        host cell. For this purpose, one can delete any of the first 20        amino acid residues at the N-terminus, particularly those at        positions 1, 2, 3, 4 and 5.    -   4. One or more glycosylation sites are removed. Residues that        are typically glycosylated (e.g., asparagine) can confer        cytolytic response. Such residues can be deleted or substituted        with unglycosylated residues (e.g., alanine).    -   5. Sites involved in interaction with complement, such as the        C1q binding site, are removed. For example, one can delete or        substitute the EKK sequence of human IgG1. Complement        recruitment may not be advantageous for the molecules of this        invention and so can be avoided with such an Fc variant.    -   6. Sites are removed that affect binding to Fc receptors other        than a salvage receptor. A native Fc can have sites for        interaction with certain white blood cells that are not required        for the fusion molecules of the present invention and so can be        removed.    -   7. The ADCC site is removed. ADCC sites are known in the art;        see, for example, Molec. Immunol. 29 (5): 633-9 (1992) with        regard to ADCC sites in IgG1. These sites, as well, are not        required for the fusion molecules of the present invention and        so can be removed.    -   8. When the native Fc is derived from a non-human antibody, the        native Fc can be humanized. Typically, to humanize a native Fc,        one will substitute selected residues in the non-human native Fc        with residues that are normally found in human native Fc.        Techniques for antibody humanization are well known in the art.

Preferred Fc variants include the following. In SEQ ID NO: 2, theleucine at position 15 can be substituted with glutamate; the glutamateat position 99, with alanine; and the lysines at positions 101 and 103,with alanines. In addition, phenyalanine residues can replace one ormore tyrosine residues.

An alternative half-life extending moiety would be a protein,polypeptide, peptide, antibody, antibody fragment, or small molecule(e.g., a peptidomimetic compound) capable of binding to a salvagereceptor. For example, one could use as a half-life extending moiety apolypeptide as described in U.S. Pat. No. 5,739,277, issued Apr. 14,1998 to Presta et al. Peptides could also be selected by phage displayfor binding to the FcRn salvage receptor. Such salvage receptor-bindingcompounds are also included within the meaning of “half-life extendingmoiety” and are within the scope of this invention. Such half-lifeextending moieties should be selected for increased half-life (e.g., byavoiding sequences recognized by proteases) and decreased immunogenicity(e.g., by favoring non-immunogenic sequences, as discovered in antibodyhumanization).

As noted above, polymer half-life extending moieties can also be usedfor F¹ and F². Various means for attaching chemical moieties useful ashalf-life extending moieties are currently available, see, e.g., PatentCooperation Treaty (“PCT”) International Publication No. WO 96/11953,entitled “N-Terminally Chemically Modified Protein Compositions andMethods,” herein incorporated by reference in its entirety. This PCTpublication discloses, among other things, the selective attachment ofwater-soluble polymers to the N-terminus of proteins.

In some embodiments of the inventive compositions, the polymer half-lifeextending moiety is polyethylene glycol (PEG), as F¹ and/or F², but itshould be understood that the inventive composition of matter, beyondpositions F¹ and/or F², can also include one or more PEGs conjugated atother sites in the molecule, such as at one or more sites on the toxinpeptide. Accordingly, some embodiments of the inventive composition ofmatter further include one or more PEG moieties conjugated to a non-PEGhalf-life extending moiety, which is F¹ and/or F², or to the toxinpeptide(s) (P), or to any combination of any of these. For example, anFc domain or portion thereof (as F1 and/or F2) in the inventivecomposition can be made mono-PEGylated, di-PEGylated, or otherwisemulti-PEGylated, by the process of reductive alkylation.

Covalent conjugation of proteins and peptides with poly(ethylene glycol)(PEG) has been widely recognized as an approach to significantly extendthe in vivo circulating half-lives of therapeutic proteins. PEGylationachieves this effect predominately by retarding renal clearance, sincethe PEG moiety adds considerable hydrodynamic radius to the protein.(Zalipsky, S., et al., Use of functionalized poly(ethylene glycol)s formodification of polypeptides., in poly(ethylene glycol) chemistry:Biotechnical and biomedical applications., J. M. Harris, Ed., PlenumPress: New York., 347-370 (1992)). Additional benefits often conferredby PEGylation of proteins and peptides include increased solubility,resistance to proteolytic degradation, and reduced immunogenicity of thetherapeutic polypeptide. The merits of protein PEGylation are evidencedby the commercialization of several PEGylated proteins includingPEG-Adenosine deaminase (Adagen™/Enzon Corp.), PEG-L-asparaginase(Oncaspar™/Enzon Corp.), PEG-Interferon α-2b(PEG-Intron™/Schering/Enzon), PEG-Interferon α-2a (PEGASYS™/Roche) andPEG-G-CSF (Neulasta™/Amgen) as well as many others in clinical trials.

Briefly, the PEG groups are generally attached to the peptide portion ofthe composition of the invention via acylation or reductive alkylation(or reductive amination) through a reactive group on the PEG moiety(e.g., an aldehyde, amino, thiol, or ester group) to a reactive group onthe inventive compound (e.g., an aldehyde, amino, or ester group).

A useful strategy for the PEGylation of synthetic peptides consists ofcombining, through forming a conjugate linkage in solution, a peptideand a PEG moiety, each bearing a special functionality that is mutuallyreactive toward the other. The peptides can be easily prepared withconventional solid phase synthesis (see, for example, FIGS. 5 and 6 andthe accompanying text herein). The peptides are “preactivated” with anappropriate functional group at a specific site. The precursors arepurified and fully characterized prior to reacting with the PEG moiety.Ligation of the peptide with PEG usually takes place in aqueous phaseand can be easily monitored by reverse phase analytical HPLC. ThePEGylated peptides can be easily purified by preparative HPLC andcharacterized by analytical HPLC, amino acid analysis and laserdesorption mass spectrometry.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). Inthe present application, the term “PEG” is used broadly to encompass anypolyethylene glycol molecule, in mono-, bi-, or poly-functional form,without regard to size or to modification at an end of the PEG, and canbe represented by the formula:X—O(CH₂CH₂O)_(n-1)CH₂CH₂OH,  (X)

where n is 20 to 2300 and X is H or a terminal modification, e.g., aC₁₋₄ alkyl.

In some useful embodiments, a PEG used in the invention terminates onone end with hydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). Itis noted that the other end of the PEG, which is shown in formula (II)terminating in OH, covalently attaches to an activating moiety via anether oxygen bond, an amine linkage, or amide linkage. When used in achemical structure, the term “PEG” includes the formula (II) abovewithout the hydrogen of the hydroxyl group shown, leaving the oxygenavailable to react with a free carbon atom of a linker to form an etherbond. More specifically, in order to conjugate PEG to a peptide, thepeptide must be reacted with PEG in an “activated” form. Activated PEGcan be represented by the formula:(PEG)-(A)  (XI)where PEG (defined supra) covalently attaches to a carbon atom of theactivation moiety (A) to form an ether bond, an amine linkage, or amidelinkage, and (A) contains a reactive group which can react with anamino, azido, alkyne, imino, maleimido, N-succinimidyl, carboxyl,aminooxy, seleno, or thiol group on an amino acid residue of a peptideor a linker moiety covalently attached to the peptide, e.g., the OSK1peptide analog. Residues baring chemoselective reactive groups can beintroduced into the toxin peptide, e.g., an OSK1 peptide analog duringassembly of the peptide sequence solid-phase synthesis as protectedderivatives. Alternatively, chemoselective reactive groups can beintroduced in the toxin peptide after assembly of the peptide sequenceby solid-phase synthesis via the use of orthogonal protecting groups atspecific sites. Examples of amino acid residues useful forchemoselective reactions include, but are not limited to,(amino-oxyacetyl)-L-diaminopropionic acid, p-azido-phenylalanine,azidohomolalanine, para-propargyloxy-phenylalanine, selenocysteine,para-acetylphenylalanine, (N^(ε)-levulinyl)-Lysine,(N^(ε)-pyruvyl)-Lysine, selenocysteine, and orthogonally protectedcysteine and homocysteine.

Accordingly, in some embodiments of the composition of matter, the toxinpeptide, e.g., the OSK1 peptide analog, is conjugated to a polyethyleneglycol (PEG) at:

(a) 1, 2, 3 or 4 amino functionalized sites in the toxin peptide;

(b) 1, 2, 3 or 4 thiol functionalized sites in the toxin peptide; (c) 1or 2 ketone functionalized sites in the toxin peptide; (d) 1 or 2 azidofunctionalized sites of the toxin peptide; (e) 1 or 2 carboxylfunctionalized sites in the toxin peptide; (f) 1 or 2 aminooxyfunctionalized sites in the toxin peptide; or (g) 1 or 2 selenofunctionalized sites in the toxin peptide.

In other embodiments of the composition of matter, the toxin peptide,e.g., the OSK1 peptide analog, is conjugated to a polyethylene glycol(PEG) at:

(a) 1, 2, 3 or 4 amino functionalized sites of the PEG;

(b) 1, 2, 3 or 4 thiol functionalized sites of the PEG;

(c) 1, 2, 3 or 4 maleimido functionalized sites of the PEG;

(d) 1, 2, 3 or 4 N-succinimidyl functionalized sites of the PEG;

(e) 1, 2, 3 or 4 carboxyl functionalized sites of the PEG; or

(f) 1, 2, 3 or 4 p-nitrophenyloxycarbonyl functionalized sites of thePEG.

Techniques for the preparation of activated PEG and its conjugation tobiologically active peptides are well known in the art. (E.g., see U.S.Pat. Nos. 5,643,575, 5,919,455, 5,932,462, and 5,990,237; Thompson etal., PEGylation of polypeptides, EP 0575545 B1; Petit, Site specificprotein modification, U.S. Pat. Nos. 6,451,986, and 6,548,644; S. Hermanet al., Poly(ethylene glycol) with reactive endgroups: I. Modificationof proteins, J. Bioactive Compatible Polymers, 10:145-187 (1995); Y. Luet al., Pegylated peptides III: Solid-phase synthesis with PEGylatingreagents of varying molecular weight: synthesis of multiply PEGylatedpeptides, Reactive Polymers, 22:221-229 (1994); A. M. Felix et al.,PEGylated Peptides IV: Enhanced biological activity of site-directedPEGylated GRF analogs, Int. J. Peptide Protein Res., 46:253-264 (1995);A. M. Felix, Site-specific poly(ethylene glycol)ylation of peptides, ACSSymposium Series 680(poly(ethylene glycol)): 218-238 (1997); Y. Ikeda etal., Polyethylene glycol derivatives, their modified peptides, methodsfor producing them and use of the modified peptides, EP 0473084 B1; G.E. Means et al., Selected techniques for the modification of proteinside chains, in: Chemical modification of proteins, Holden Day, Inc.,219 (1971)).

Activated PEG, such as PEG-aldehydes or PEG-aldehyde hydrates, can bechemically synthesized by known means or obtained from commercialsources, e.g., Shearwater Polymers, (Huntsville, Ala.) or Enzon, Inc.(Piscataway, N.J.).

An example of a useful activated PEG for purposes of the presentinvention is a PEG-aldehyde compound (e.g., a methoxy PEG-aldehyde),such as PEG-propionaldehyde, which is commercially available fromShearwater Polymers (Huntsville, Ala.). PEG-propionaldehyde isrepresented by the formula PEG-CH₂CH₂CHO. (See, e.g., U.S. Pat. No.5,252,714). Other examples of useful activated PEG are PEG acetaldehydehydrate and PEG bis aldehyde hydrate, which latter yields abifunctionally activated structure. (See., e.g., Bentley et al.,Poly(ethylene glycol) aldehyde hydrates and related polymers andapplications in modifying amines, U.S. Pat. No. 5,990,237).

Another useful activated PEG for generating the PEG-conjugated peptidesof the present invention is a PEG-maleimide compound, such as, but notlimited to, a methoxy PEG-maleimide, such as maleimido monomethoxy PEG,are particularly useful for generating the PEG-conjugated peptides ofthe invention. (E.g., Shen, N-maleimidyl polymer derivatives, U.S. Pat.No. 6,602,498; C. Delgado et al., The uses and properties of PEG-linkedproteins., Crit. Rev. Therap. Drug Carrier Systems, 9:249-304 (1992); S.Zalipsky et al., Use of functionalized poly(ethylene glycol)s formodification of polypeptides, in: Poly(ethylene glycol) chemistry:Biotechnical and biomedical applications (J. M. Harris, Editor, PlenumPress: New York, 347-370 (1992); S. Herman et al., Poly(ethylene glycol)with reactive endgroups: I. Modification of proteins, J. BioactiveCompatible Polymers, 10:145-187 (1995); P. J. Shadle et al., Conjugationof polymer to colony stimulating factor-1, U.S. Pat. No. 4,847,325; G.Shaw et al., Cysteine added variants IL-3 and chemical modificationsthereof, U.S. Pat. No. 5,166,322 and EP 0469074 B1; G. Shaw et al.,Cysteine added variants of EPO and chemical modifications thereof, EP0668353 A1; G. Shaw et al., Cysteine added variants G-CSF and chemicalmodifications thereof, EP 0668354 A1; N. V. Katre et al., Interleukin-2muteins and polymer conjugation thereof, U.S. Pat. No. 5,206,344; R. J.Goodson and N. V. Katre, Site-directed pegylation of recombinantinterleukin-2 at its glycosylation site, Biotechnology, 8:343-346(1990)).

A poly(ethylene glycol) vinyl sulfone is another useful activated PEGfor generating the PEG-conjugated peptides of the present invention byconjugation at thiolated amino acid residues, e.g., at C residues.(E.g., M. Morpurgo et al., Preparation and characterization ofpoly(ethylene glycol) vinyl sulfone, Bioconj. Chem., 7:363-368 (1996);see also Harris, Functionalization of polyethylene glycol for formationof active sulfone-terminated PEG derivatives for binding to proteins andbiologically compatible materials, U.S. Pat. Nos. 5,446,090; 5,739,208;5,900,461; 6,610,281 and 6,894,025; and Harris, Water soluble activesulfones of poly(ethylene glycol), WO 95/13312 A1).

Another activated form of PEG that is useful in accordance with thepresent invention, is a PEG-N-hydroxysuccinimide ester compound, forexample, methoxy PEG-N-hydroxysuccinimidyl (NHS) ester.

Heterobifunctionally activated forms of PEG are also useful. (See, e.g.,Thompson et al., PEGylation reagents and biologically active compoundsformed therewith, U.S. Pat. No. 6,552,170).

Typically, a toxin peptide or, a fusion protein comprising the toxinpeptide, is reacted by known chemical techniques with an activated PEGcompound, such as but not limited to, a thiol-activated PEG compound, adiol-activated PEG compound, a PEG-hydrazide compound, a PEG-oxyaminecompound, or a PEG-bromoacetyl compound. (See, e.g., S. Herman,Poly(ethylene glycol) with Reactive Endgroups: I. Modification ofProteins, J. Bioactive and Compatible Polymers, 10:145-187 (1995); S.Zalipsky, Chemistry of Polyethylene Glycol Conjugates with BiologicallyActive Molecules, Advanced Drug Delivery Reviews, 16:157-182 (1995); R.Greenwald et al., Poly(ethylene glycol) conjugated drugs and prodrugs: acomprehensive review, Critical Reviews in Therapeutic Drug CarrierSystems, 17:101-161 (2000)).

Methods for N-terminal PEGylation are exemplified herein in Examples31-34, 45 and 47-48, but these are in no way limiting of the PEGylationmethods that can be employed by one skilled in the art.

Any molecular mass for a PEG can be used as practically desired, e.g.,from about 1,000 or 2,000 Daltons (Da) to about 100,000 Da (n is 20 to2300). Preferably, the combined or total molecular mass of PEG used in aPEG-conjugated peptide of the present invention is from about 3,000 Daor 5,000 Da, to about 50,000 Da or 60,000 Da (total n is from 70 to1,400), more preferably from about 10,000 Da to about 40,000 Da (total nis about 230 to about 910). The most preferred combined mass for PEG isfrom about 20,000 Da to about 30,000 Da (total n is about 450 to about680). The number of repeating units “n” in the PEG is approximated forthe molecular mass described in Daltons. It is preferred that thecombined molecular mass of PEG on an activated linker is suitable forpharmaceutical use. Thus, the combined molecular mass of the PEGmolecule should not exceed about 100,000 Da.

Polysaccharide polymers are another type of water-soluble polymer thatcan be used for protein modification. Dextrans are polysaccharidepolymers comprised of individual subunits of glucose predominantlylinked by α1-6 linkages. The dextran itself is available in manymolecular weight ranges, and is readily available in molecular weightsfrom about 1 kDa to about 70 kDa. Dextran is a suitable water-solublepolymer for use in the present invention as a half-life extending moietyby itself or in combination with another half-life extending moiety(e.g., Fc). See, for example, WO 96/11953 and WO 96/05309. The use ofdextran conjugated to therapeutic or diagnostic immunoglobulins has beenreported; see, for example, European Patent Publication No. 0 315 456,which is hereby incorporated by reference in its entirety. Dextran ofabout 1 kDa to about 20 kDa is preferred when dextran is used as ahalf-life extending moiety in accordance with the present invention.

Linkers. Any “linker” group or moiety (i.e., “-(L)_(f)-” or “-(L)_(g)-”in Formulae I-IX) is optional. When present, its chemical structure isnot critical, since it serves primarily as a spacer. As stated hereinabove, the linker moiety (-(L)_(f)- and/or -(L)_(g)-), if present, canbe independently the same or different from any other linker, orlinkers, that may be present in the inventive composition. For example,an “(L)_(f)” can represent the same moiety as, or a different moietyfrom, any other “(L)_(f)” or any “(L)_(g)” in accordance with theinvention. The linker is preferably made up of amino acids linkedtogether by peptide bonds. Some of these amino acids can beglycosylated, as is well understood by those in the art. For example, auseful linker sequence constituting a sialylation site is X₁X₂NX₄X₅G(SEQ ID NO: 637), wherein X₁, X₂, X₄ and X₅ are each independently anyamino acid residue.

As stated above, in some embodiments, a peptidyl linker is present(i.e., made up of amino acids linked together by peptide bonds) that ismade in length, preferably, of from 1 up to about 40 amino acidresidues, more preferably, of from 1 up to about 20 amino acid residues,and most preferably of from 1 to about 10 amino acid residues.Preferably, but not necessarily, the amino acid residues in the linkerare from among the twenty canonical amino acids, more preferably,cysteine, glycine, alanine, proline, asparagine, glutamine, and/orserine. Even more preferably, a peptidyl linker is made up of a majorityof amino acids that are sterically unhindered, such as glycine, serine,and alanine linked by a peptide bond. It is also desirable that, ifpresent, a peptidyl linker be selected that avoids rapid proteolyticturnover in circulation in vivo. Thus, preferred linkers includepolyglycines (particularly (Gly)₄ (SEQ ID NO: 4918), (Gly)₅) (SEQ ID NO:4919), poly(Gly-Ala), and polyalanines. Other preferred linkers arethose identified herein as “L5” (GGGGS; SEQ ID NO: 638), “L10”(GGGGSGGGGS; SEQ ID NO:79), “L25” GGGGSGGGGSGGGGSGGGGSGGGGS; SEQ IDNO:84) and any linkers used in the working examples hereinafter. Thelinkers described herein, however, are exemplary; linkers within thescope of this invention can be much longer and can include otherresidues.

In some embodiments of the compositions of this invention, whichcomprise a peptide linker moiety (L), acidic residues, for example,glutamate or aspartate residues, are placed in the amino acid sequenceof the linker moiety (L). Examples include the following peptide linkersequences:

GGEGGG; (SEQ ID NO: 639) GGEEEGGG; (SEQ ID NO: 640) GEEEG;(SEQ ID NO: 641) GEEE; (SEQ ID NO: 642) GGDGGG; (SEQ ID NO: 643)GGDDDGG; (SEQ ID NO: 644) GDDDG; (SEQ ID NO: 645) GDDD; (SEQ ID NO: 646)GGGGSDDSDEGSDGEDGGGGS; (SEQ ID NO: 647) WEWEW; (SEQ ID NO: 648) FEFEF;(SEQ ID NO: 649) EEEWWW; (SEQ ID NO: 650) EEEFFF; (SEQ ID NO: 651)WWEEEWW; (SEQ ID NO: 652) or FFEEEFF. (SEQ ID NO: 653)

In other embodiments, the linker constitutes a phosphorylation site,e.g., X₁X₂YX₃X₄G (SEQ ID NO: 654), wherein X₁, X₂, X₃ and X₄ are eachindependently any amino acid residue; X₁X₂SX₃X₄G (SEQ ID NO: 655),wherein X₁, X₂, X₃ and X₄ are each independently any amino acid residue;or X₁X₂TX₃X₄G (SEQ ID NO: 656), wherein X₁, X₂, X₃ and X₄ are eachindependently any amino acid residue.

Non-peptide linkers are also possible. For example, alkyl linkers suchas —NH—(CH₂)_(s)—C(O)—, wherein s=2-20 could be used. These alkyllinkers can further be substituted by any non-sterically hindering groupsuch as lower alkyl (e.g., C₁-C₆) lower acyl, halogen (e.g., Cl, Br),CN, NH₂, phenyl, etc. An exemplary non-peptide linker is a PEG linker,

wherein n is such that the linker has a molecular weight of 100 to 5000kDa, preferably 100 to 500 kDa. The peptide linkers can be altered toform derivatives in the same manner as described above.

Useful linker embodiments also include aminoethyloxyethyloxy-acetyllinkers as disclosed by Chandy et al. (Chandy et al., WO 2006/042151 A2,incorporated herein by reference in its entirety).

Derivatives. The inventors also contemplate derivatizing the peptideand/or half-life extending moiety portion of the compounds. Suchderivatives can improve the solubility, absorption, biologicalhalf-life, and the like of the compounds. The moieties can alternativelyeliminate or attenuate any undesirable side-effect of the compounds andthe like. Exemplary derivatives include compounds in which:

-   1. The compound or some portion thereof is cyclic. For example, the    peptide portion can be modified to contain two or more Cys residues    (e.g., in the linker), which could cyclize by disulfide bond    formation.-   2. The compound is cross-linked or is rendered capable of    cross-linking between molecules. For example, the peptide portion    can be modified to contain one Cys residue and thereby be able to    form an intermolecular disulfide bond with a like molecule. The    compound can also be cross-linked through its C-terminus, as in the    molecule shown below.

-   3. Non-peptidyl linkages (bonds) replace one or more peptidyl    [—C(O)NR—] linkages. Exemplary non-peptidyl linkages are    —CH₂-carbamate [—CH₂—OC(O)NR—], phosphonate, —CH₂-sulfonamide    [—CH₂—S(O)₂NR—], urea [—NHC(O)NH—], —CH₂-secondary amine, and    alkylated peptide [—C(O)NR⁶— wherein R⁶ is lower alkyl].-   4. The N-terminus is chemically derivatized. Typically, the    N-terminus can be acylated or modified to a substituted amine.    Exemplary N-terminal derivative groups include —NRR¹ (other than    —NH₂), —NRC(O)R¹, —NRC(O)OR¹, —NRS(O)₂R¹, —NHC(O)NHR¹, succinimide,    or benzyloxycarbonyl-NH— (CBZ-NH—), wherein R and R¹ are each    independently hydrogen or lower alkyl and wherein the phenyl ring    can be substituted with 1 to 3 substituents selected from the group    consisting of C₁-C₄ alkyl, C₁-C₄ alkoxy, chloro, and bromo.-   5. The free C-terminus is derivatized. Typically, the C-terminus is    esterified or amidated. For example, one can use methods described    in the art to add (NH—CH₂—CH₂—NH₂)₂ to compounds of this invention    having any of SEQ ID NOS: 504 to 508 at the C-terminus. Likewise,    one can use methods described in the art to add —NH₂ to compounds of    this invention having any of SEQ ID NOS: 924 to 955, 963 to 972,    1005 to 1013, or 1018 to 1023 at the C-terminus. Exemplary    C-terminal derivative groups include, for example, —C(O)R² wherein    R² is lower alkoxy or —NR³R⁴ wherein R³ and R⁴ are independently    hydrogen or C₁-C₈ alkyl (preferably C₁-C₄ alkyl).-   6. A disulfide bond is replaced with another, preferably more    stable, cross-linking moiety (e.g., an alkylene). See, e.g.,    Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9; Alberts et    al. (1993) Thirteenth Am. Pep. Symp., 357-9.-   7. One or more individual amino acid residues are modified. Various    derivatizing agents are known to react specifically with selected    sidechains or terminal residues, as described in detail below.

Lysinyl residues and amino terminal residues can be reacted withsuccinic or other carboxylic acid anhydrides, which reverse the chargeof the lysinyl residues. Other suitable reagents for derivatizingalpha-amino-containing residues include imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride;trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; andtransaminase-catalyzed reaction with glyoxylate.

Arginyl residues can be modified by reaction with any one or combinationof several conventional reagents, including phenylglyoxal,2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization ofarginyl residues requires that the reaction be performed in alkalineconditions because of the high pKa of the guanidine functional group.Furthermore, these reagents can react with the groups of lysine as wellas the arginine epsilon-amino group.

Specific modification of tyrosyl residues has been studied extensively,with particular interest in introducing spectral labels into tyrosylresidues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizole andtetranitromethane are used to form O-acetyl tyrosyl species and 3-nitroderivatives, respectively.

Carboxyl sidechain groups (aspartyl or glutamyl) can be selectivelymodified by reaction with carbodiimides (R′—N═C═N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues can be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues can be deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Cysteinyl residues can be replaced by amino acid residues or othermoieties either to eliminate disulfide bonding or, conversely, tostabilize cross-linking. See, e.g., Bhatnagar et al. (1996), J. Med.Chem. 39: 3814-9.

Derivatization with bifunctional agents is useful for cross-linking thepeptides or their functional derivatives to a water-insoluble supportmatrix or to other macromolecular half-life extending moieties. Commonlyused cross-linking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Carbohydrate (oligosaccharide) groups can conveniently be attached tosites that are known to be glycosylation sites in proteins. Generally,O-linked oligosaccharides are attached to serine (Ser) or threonine(Thr) residues while N-linked oligosaccharides are attached toasparagine (Asn) residues when they are part of the sequenceAsn-X-Ser/Thr, where X can be any amino acid except proline. X ispreferably one of the 19 naturally occurring amino acids other thanproline. The structures of N-linked and O-linked oligosaccharides andthe sugar residues found in each type are different. One type of sugarthat is commonly found on both is N-acetylneuraminic acid (referred toas sialic acid). Sialic acid is usually the terminal residue of bothN-linked and O-linked oligosaccharides and, by virtue of its negativecharge, can confer acidic properties to the glycosylated compound. Suchsite(s) can be incorporated in the linker of the compounds of thisinvention and are preferably glycosylated by a cell during recombinantproduction of the polypeptide compounds (e.g., in mammalian cells suchas CHO, BHK, COS). However, such sites can further be glycosylated bysynthetic or semi-synthetic procedures known in the art.

Other possible modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, oxidation of the sulfur atom in Cys, methylation of thealpha-amino groups of lysine, arginine, and histidine side chains.Creighton, Proteins: Structure and Molecule Properties (W. H. Freemanand Co., San Francisco), pp. 79-86 (1983).

Compounds of the present invention can be changed at the DNA level, aswell. The DNA sequence of any portion of the compound can be changed tocodons more compatible with the chosen host cell. For E. coli, which isthe preferred host cell, optimized codons are known in the art. Codonscan be substituted to eliminate restriction sites or to include silentrestriction sites, which can aid in processing of the DNA in theselected host cell. The half-life extending moiety, linker and peptideDNA sequences can be modified to include any of the foregoing sequencechanges.

A process for preparing conjugation derivatives is also contemplated.Tumor cells, for example, exhibit epitopes not found on their normalcounterparts. Such epitopes include, for example, differentpost-translational modifications resulting from their rapidproliferation. Thus, one aspect of this invention is a processcomprising:

-   -   a) selecting at least one randomized peptide that specifically        binds to a target epitope; and    -   b) preparing a pharmacologic agent comprising (i) at least one        half-life extending moiety (Fc domain preferred), (ii) at least        one amino acid sequence of the selected peptide or peptides,        and (iii) an effector molecule.        The target epitope is preferably a tumor-specific epitope or an        epitope specific to a pathogenic organism. The effector molecule        can be any of the above-noted conjugation partners and is        preferably a radioisotope.

Methods of Making

The present invention also relates to nucleic acids, expression vectorsand host cells useful in producing the polypeptides of the presentinvention. Host cells can be eukaryotic cells, with mammalian cellspreferred and CHO cells most preferred. Host cells can also beprokaryotic cells, with E. coli cells most preferred.

The compounds of this invention largely can be made in transformed hostcells using recombinant DNA techniques. To do so, a recombinant DNAmolecule coding for the peptide is prepared. Methods of preparing suchDNA molecules are well known in the art. For instance, sequences codingfor the peptides could be excised from DNA using suitable restrictionenzymes. Alternatively, the DNA molecule could be synthesized usingchemical synthesis techniques, such as the phosphoramidate method. Also,a combination of these techniques could be used.

The invention also includes a vector capable of expressing the peptidesin an appropriate host. The vector comprises the DNA molecule that codesfor the peptides operatively linked to appropriate expression controlsequences. Methods of effecting this operative linking, either before orafter the DNA molecule is inserted into the vector, are well known.Expression control sequences include promoters, activators, enhancers,operators, ribosomal binding sites, start signals, stop signals, capsignals, polyadenylation signals, and other signals involved with thecontrol of transcription or translation.

The resulting vector having the DNA molecule thereon is used totransform an appropriate host. This transformation can be performedusing methods well known in the art.

Any of a large number of available and well-known host cells can be usedin the practice of this invention. The selection of a particular host isdependent upon a number of factors recognized by the art. These include,for example, compatibility with the chosen expression vector, toxicityof the peptides encoded by the DNA molecule, rate of transformation,ease of recovery of the peptides, expression characteristics, bio-safetyand costs. A balance of these factors must be struck with theunderstanding that not all hosts can be equally effective for theexpression of a particular DNA sequence. Within these generalguidelines, useful microbial hosts include bacteria (such as E. colisp.), yeast (such as Saccharomyces sp.) and other fungi, insects,plants, mammalian (including human) cells in culture, or other hostsknown in the art.

Next, the transformed host is cultured and purified. Host cells can becultured under conventional fermentation conditions so that the desiredcompounds are expressed. Such fermentation conditions are well known inthe art. Finally, the peptides are purified from culture by methods wellknown in the art.

In some embodiments of the inventive DNA, the DNA encodes a recombinantfusion protein composition of the invention, preferably, but notnecessarily, monovalent with respect to the toxin peptide, forexpression in a mammalian cell, such as, but not limited to, CHO orHEK293. The encoded fusion protein includes (a)-(c) immediately below,in the N-terminal to C-terminal direction:

(a) an immunoglobulin, which includes the constant and variable regionsof the immunoglobulin light and heavy chains, or a portion of animmunoglobulin (e.g., an Fc domain, or the variable regions of the lightand heavy chains); if both immunoglobulin light chain and heavy chaincomponents are to be included in the construct, then a peptidyl linker,as further described in (b) immediately below, is also included toseparate the immunoglobulin components (See, e.g., FIG. 92A-C); usefulcoding sequences for immunoglobulin light and heavy chains are wellknown in the art;

(b) a peptidyl linker, which is at least 4 (or 5) amino acid residueslong and comprises at least one protease cleavage site (e.g., a furincleavage site, which is particularly useful for intracellular cleavageof the expressed fusion protein); typically, the peptidyl linkersequence can be up to about 35 to 45 amino acid residues long (e.g., a7x L5 linker modified to include the desired protease cleavage site(s)),but linkers up to about 100 to about 300 amino acid residues long arealso useful; and

(c) an immunoglobulin Fc domain or a portion thereof. The Fc domain of(c) can be from the same type of immunoglobulin in (a), or different. Insuch embodiments, the DNA encodes a toxin peptide covalently linked tothe N-terminal or C-terminal end of (a) or (c) above, either directly orindirectly via a peptidyl linker (a linker minus a protease cleavagesite). Any toxin peptide or peptide analog thereof as described hereincan be encoded by the DNA (e.g., but not limited to, ShK, HmK, MgTx,AgTx1, AgTx2, HsTx1, OSK1, Anuroctoxin, Noxiustoxin, Hongotoxin, HsTx1,ChTx, MTx, Titystoxin, BgK, BmKTX, BmTx, Tc30, Tc32, Pi1, Pi2, Pi3 toxinpeptide, or a peptide analog of any of these). For example, an OSK1peptide analog comprising an amino acid sequence selected from SEQ IDNOS: 25, 294 through 298, 562 through 636, 980 through 1274, 1303, 1308,1391 through 4912, 4916, 4920 through 5006, 5009, 5010, and 5012 through5015, as set forth in Tables 7 and Tables 7A-J, can be employed.Alternatively, an ShK peptide analog comprising an amino acid sequenceselected from SEQ ID NOS: 5, 88 through 200, 548 through 561, 884through 950, and 1295 through 1300 as set forth in Table 2, can beemployed. Any other toxin peptide sequence described herein that canalternatively be expressed recombinantly using recombinant and proteinengineering techniques known in the art can also be used. Theimmunoglobulin of (a) and (c) above can be in each instanceindependently selected from any desired type, such as but not limitedto, IgG1, IgG2, IgG3, and IgG4. The variable regions can benon-functional in vivo (e.g., CDRs specifically binding KLH), oralternatively, if targeting enhancement function is also desired, thevariable regions can be chosen to specifically bind (non-competitively)the ion channel target of the toxin peptide (e.g., Kv1.3) orspecifically bind another antigen typically found associated with, or inthe vicinity of, the target ion channel. In addition, the inventive DNAoptionally further encodes, 5′ to the coding region of (a) above, asignal peptide sequence (e.g., a secretory signal peptide) operablylinked to the expressed fusion protein. An example of the inventive DNAencoding a recombinant fusion protein for expression in a mammaliancell, described immediately above, is a DNA that encodes a fusionprotein comprising, in the N-terminal to C-terminal direction:

(a) an immunoglobulin light chain;

(b) a first peptidyl linker at least 4 amino acid residues longcomprising at least one protease cleavage site, as described above;

(c) an immunoglobulin heavy chain;

(d) a second peptidyl linker at least 4 amino acid residues longcomprising at least one protease cleavage site, as described above; and

(e) an immunoglobulin Fc domain or a portion thereof. Here, the Fcdomain of (e) can be from the same type of immunoglobulin as the heavychain in (c), or different. The DNA encodes a toxin peptide covalentlylinked to the N-terminal or C-terminal end of (a), (c), or (e) of theexpressed fusion protein, either directly or indirectly via a peptidyllinker (a linker minus a protease cleavage site). FIG. 92A-C illustratesschematically an embodiment, in which the toxin peptide (e.g., an OSK1,ShK, or a peptide analog of either of these) is covalently linked to theC-terminal end of the Fc domain of (e). In FIG. 92A-C, a linker is showncovalently linking the toxin peptide to the rest of the molecule, but aspreviously described, this linker is optional.

In some embodiments particularly suited for the recombinant expressionof monovalent dimeric Fc-toxin peptide fusions or “peptibodies” (see,FIG. 2B and Example 56) by mammalian cells, such as, but not limited to,CHO or HEK293, the inventive DNA encodes a recombinant expressed fusionprotein that comprises, in the N-terminal to C-terminal direction:

-   -   (a) a first immunoglobulin Fc domain or portion thereof;    -   (b) a peptidyl linker at least 4 (or 5) amino acid residues long        comprising at least one protease cleavage site (e.g., a furin        cleavage site, which is particularly useful for intracellular        cleavage of the expressed fusion protein); typically, the        peptidyl linker sequence can be up to about 35 to 45 amino acid        residues long (e.g., a 7x L5 linker modified to include the        desired protease cleavage site(s)), but linkers up to about 100        to about 300 amino acid residues long are also useful; and    -   (c) a second immunoglobulin Fc domain or portion thereof (which        may be the same or different from the first Fc domain, but        should be expressed in the same orientation as the first Fc        domain).        For such embodiments, the DNA encodes a toxin peptide covalently        linked to the N-terminal or C-terminal end of (a) or (c) of the        expressed fusion protein, either directly or indirectly via a        peptidyl linker (a linker minus a protease cleavage site);        Example 56 describes an embodiment in which the toxin peptide is        conjugated to the C-terminal end of the second immunoglobulin Fc        domain (c). Any toxin peptide or peptide analog thereof as        described herein can be encoded by the DNA (e.g., but not        limited to, ShK, HmK, MgTx, AgTx1, AgTx2, HsTx1, OSK1,        Anuroctoxin, Noxiustoxin, Hongotoxin, HsTx1, ChTx, MTx,        Titystoxin, BgK, BmKTX, BmTx, Tc30, Tc32, Pi1, Pi2, Pi3 toxin        peptide, or a peptide analog of any of these). For example, an        OSK1 peptide analog comprising an amino acid sequence selected        from SEQ ID NOS: 25, 294 through 298, 562 through 636, 980        through 1274, 1303, 1308, 1391 through 4912, 4916, 4920 through        5006, 5009, 5010, and 5012 through 5015, as set forth in Tables        7 and Tables 7A-J, can be employed. Alternatively, an ShK        peptide analog comprising an amino acid sequence selected from        SEQ ID NOS: 5, 88 through 200, 548 through 561, 884 through 950,        and 1295 through 1300 as set forth in Table 2, can be employed.        Any other toxin peptide sequence described herein that can        alternatively be expressed using recombinant and protein        engineering techniques known in the art can also be used. In        addition, the inventive DNA optionally further encodes, 5′ to        the coding region of (a) above, a signal peptide sequence (e.g.,        a secretory signal peptide) operably linked to the expressed        fusion protein.

DNA constructs similar to those described above are also useful forrecombinant expression by mammalian cells of other dimeric Fc fusionproteins (“peptibodies”) or chimeric immunoglobulin (light chain+heavychain)-Fc heterotrimers (“hemibodies”), conjugated to pharmacologicallyactive peptides (e.g., agonist or antagonist peptides) other than toxinpeptides.

Peptide compositions of the present invention can also be made bysynthetic methods. Solid phase synthesis is the preferred technique ofmaking individual peptides since it is the most cost-effective method ofmaking small peptides. For example, well known solid phase synthesistechniques include the use of protecting groups, linkers, and solidphase supports, as well as specific protection and deprotection reactionconditions, linker cleavage conditions, use of scavengers, and otheraspects of solid phase peptide synthesis. Suitable techniques are wellknown in the art. (E.g., Merrifield (1973), Chem. Polypeptides, pp.335-61 (Katsoyannis and Panayotis eds.); Merrifield (1963), J. Am. Chem.Soc. 85: 2149; Davis et al. (1985), Biochem. Intl. 10: 394-414; Stewartand Young (1969), Solid Phase Peptide Synthesis; U.S. Pat. No.3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2: 105-253; andErickson et al. (1976), The Proteins (3rd ed.) 2: 257-527; “ProtectingGroups in Organic Synthesis,” 3rd Edition, T. W. Greene and P. G. M.Wuts, Eds., John Wiley & Sons, Inc., 1999; NovaBiochem Catalog, 2000;“Synthetic Peptides, A User's Guide,” G. A. Grant, Ed., W.H. Freeman &Company, New York, N.Y., 1992; “Advanced Chemtech Handbook ofCombinatorial & Solid Phase Organic Chemistry,” W. D. Bennet, J. W.Christensen, L. K. Hamaker, M. L. Peterson, M. R. Rhodes, and H. H.Saneii, Eds., Advanced Chemtech, 1998; “Principles of Peptide Synthesis,2nd ed.,” M. Bodanszky, Ed., Springer-Verlag, 1993; “The Practice ofPeptide Synthesis, 2nd ed.,” M. Bodanszky and A. Bodanszky, Eds.,Springer-Verlag, 1994; “Protecting Groups,” P. J. Kocienski, Ed., GeorgThieme Verlag, Stuttgart, Germany, 1994; “Fmoc Solid Phase PeptideSynthesis, A Practical Approach,” W. C. Chan and P. D. White, Eds.,Oxford Press, 2000, G. B. Fields et al., Synthetic Peptides: A User'sGuide, 1990, 77-183).

Whether the compositions of the present invention are prepared bysynthetic or recombinant techniques, suitable protein purificationtechniques can also be involved, when applicable. In some embodiments ofthe compositions of the invention, the toxin peptide portion and/or thehalf-life extending portion, or any other portion, can be prepared toinclude a suitable isotopic label (e.g., ¹²⁵I, ¹⁴C, ¹³C, ³⁵S, ³H, ²H,¹³N, ¹⁵N, ¹⁸O, ¹⁷O, etc.), for ease of quantification or detection.

Compounds that contain derivatized peptides or which contain non-peptidegroups can be synthesized by well-known organic chemistry techniques.

Uses of the Compounds

In general. The compounds of this invention have pharmacologic activityresulting from their ability to bind to proteins of interest asagonists, mimetics or antagonists of the native ligands of such proteinsof interest. Heritable diseases that have a known linkage to ionchannels (“channelopathies”) cover various fields of medicine, some ofwhich include neurology, nephrology, myology and cardiology. A list ofinherited disorders attributed to ion channels includes:

-   -   cystic fibrosis (Cl⁻ channel; CFTR),    -   Dent's disease (proteinuria and hypercalciuria; Cl⁻ channel;        CLCN5),    -   osteopetrosis (Cl⁻ channel; CLCN7),    -   familial hyperinsulinemia (SUR1; KCNJ11; K channel),    -   diabetes (KATP/SUR channel),    -   Andersen syndrome (KCNJ2, Kir2.1 K channel),    -   Bartter syndrome (KCNJ1; Kir1.1/ROMK; K channel),    -   hereditary hearing loss (KCNQ4; K channel),    -   hereditary hypertension (Liddle's syndrome; SCNN1; epithelial Na        channel),    -   dilated cardiomyopathy (SUR2, K channel),    -   long-QT syndrome or cardiac arrhythmias (cardiac potassium and        sodium channels),    -   Thymothy syndrome (CACNA1C, Cav1.2),    -   myasthenic syndromes (CHRNA, CHRNB, CNRNE; nAChR), and a variety        of other myopathies,    -   hyperkalemic periodic paralysis (Na and K channels),    -   epilepsy (Na⁺ and K⁺ channels),    -   hemiplegic migraine (CACNA1A, Cav2.1 Ca²⁺ channel and ATP1A2),    -   central core disease (RYR1, RYR1; Ca²⁺ channel), and    -   paramyotonia and myotonia (Na⁺, Cl⁻ channels)        See L. J. Ptacek and Y—H Fu (2004), Arch. Neurol. 61:        166-8; B. A. Niemeyer et al. (2001), EMBO reports 21: 568-73; F.        Lehmann-Horn and K. Jurkat-Rott (1999), Physiol. Rev. 79:        1317-72. Although the foregoing list concerned disorders of        inherited origin, molecules targeting the channels cited in        these disorders can also be useful in treating related disorders        of other, or indeterminate, origin.

In addition to the aforementioned disorders, evidence has also beenprovided supporting ion channels as targets for treatment of:

-   -   sickle cell anemia (IKCa1)—in sickle cell anemia, water loss        from erythrocytes leads to hemoglobin polymerization and        subsequent hemolysis and vascular obstruction. The water loss is        consequent to potassium efflux through the so-called Gardos        channel i.e., IKCa1. Therefore, block of IKCa1 is a potential        therapeutic treatment for sickle cell anemia.    -   glaucoma (BKCa),—in glaucoma the intraocular pressure is too        high leading to optic nerve damage, abnormal eye function and        possibly blindness. Block of BKCa potassium channels can reduce        intraocular fluid secretion and increase smooth muscle        contraction, possibly leading to lower intraocular pressure and        neuroprotection in the eye.    -   multiple sclerosis (Kv, KCa),    -   psoriasis (Kv, KCa),    -   arthritis (Kv, KCa),    -   asthma (KCa, Kv),    -   allergy (KCa, Kv),    -   COPD (KCa, Kv, Ca),    -   allergic rhinitis (KCa, Kv),    -   pulmonary fibrosis,    -   lupus (IKCa1, Kv),    -   transplantation, GvHD (KCa, Kv),    -   inflammatory bone resorption (KCa, Kv),    -   periodontal disease (KCa, Kv),    -   diabetes, type I (Kv),—type I diabetes is an autoimmune disease        that is characterized by abnormal glucose, protein and lipid        metabolism and is associated with insulin deficiency or        resistance. In this disease, Kv1.3-expressing T-lymphocytes        attack and destroy pancreatic islets leading to loss of        beta-cells. Block of Kv1.3 decreases inflammatory cytokines. In        addition block of Kv1.3 facilitates the translocation of GLUT4        to the plasma membrane, thereby increasing insulin sensitivity.    -   obesity (Kv),—Kv1.3 appears to play a critical role in        controlling energy homeostasis and in protecting against        diet-induced obesity. Consequently, Kv1.3 blockers could        increase metabolic rate, leading to greater energy utilization        and decreased body weight.    -   restenosis (KCa, Ca²⁺),—proliferation and migration of vascular        smooth muscle cells can lead to neointimal thickening and        vascular restenosis. Excessive neointimal vascular smooth muscle        cell proliferation is associated with elevated expression of        IKCa1. Therefore, block of IKCa1 could represent a therapeutic        strategy to prevent restenosis after angioplasty.    -   ischaemia (KCa, Ca²⁺),—in neuronal or cardiac ischemia,        depolarization of cell membranes leads to opening of        voltage-gated sodium and calcium channels. In turn this can lead        to calcium overload, which is cytotoxic. Block of voltage-gated        sodium and/or calcium channels can reduce calcium overload and        provide cytoprotective effects. In addition, due to their        critical role in controlling and stabilizing cell membrane        potential, modulators of voltage- and calcium-activated        potassium channels can also act to reduce calcium overload and        protect cells.    -   renal incontinence (KCa), renal incontinence is associated with        overactive bladder smooth muscle cells. Calcium-activated        potassium channels are expressed in bladder smooth muscle cells,        where they control the membrane potential and indirectly control        the force and frequency of cell contraction. Openers of        calcium-activated potassium channels therefore provide a        mechanism to dampen electrical and contractile activity in        bladder, leading to reduced urge to urinate.    -   osteoporosis (Kv),    -   pain, including migraine (Na_(v), TRP [transient receptor        potential channels], P2X, Ca²⁺), N-type voltage-gated calcium        channels are key regulators of nociceptive neurotransmission in        the spinal cord. Ziconotide, a peptide blocker of N-type calcium        channels reduces nociceptive neurotransmission and is approved        worldwide for the symptomatic alleviation of severe chronic pain        in humans. Novel blockers of nociceptor-specific N-type calcium        channels would be improved analgesics with reduced side-effect        profiles.    -   hypertension (Ca²⁺),—L-type and T-type voltage-gated calcium        channels are expressed in vascular smooth muscle cells where        they control excitation-contraction coupling and cellular        proliferation. In particular, T-type calcium channel activity        has been linked to neointima formation during hypertension.        Blockers of L-type and T-type calcium channels are useful for        the clinical treatment of hypertension because they reduce        calcium influx and inhibit smooth muscle cell contraction.    -   wound healing, cell migration serves a key role in wound        healing. Intracellular calcium gradients have been implicated as        important regulators of cellular migration machinery in        keratinocytes and fibroblasts. In addition, ion flux across cell        membranes is associated with cell volume changes. By controlling        cell volume, ion channels contribute to the intracellular        environment that is required for operation of the cellular        migration machinery. In particular, IKCa1 appears to be required        universally for cell migration. In addition, Kv1.3, Kv3.1, NMDA        receptors and N-type calcium channels are associated with the        migration of lymphocytes and neurons.    -   stroke,    -   Alzheimer's,    -   Parkenson's Disease (nACHR, Nav)    -   Bipolar Disorder (Nav, Cav)    -   cancer, many potassium channel genes are amplified and protein        subunits are upregulated in many cancerous condition. Consistent        with a pathophysiological role for potassium channel        upregulation, potassium channel blockers have been shown to        suppress proliferation of uterine cancer cells and        hepatocarcinoma cells, presumably through inhibition of calcium        influx and effects on calcium-dependent gene expression.    -   a variety of neurological, cardiovascular, metabolic and        autoimmune diseases.

Both agonists and antagonists of ion channels can achieve therapeuticbenefit. Therapeutic benefits can result, for example, from antagonizingKv1.3, IKCa1, SKCa, BKCa, N-type or T-type Ca²⁺ channels and the like.Small molecule and peptide antagonists of these channels have been shownto possess utility in vitro and in vivo. Limitations in productionefficiency and pharmacokinetics, however, have largely preventedclinical investigation of inhibitor peptides of ion channels.

Compositions of this invention incorporating peptide antagonists of thevoltage-gated potassium channel Kv1.3, in particular OSK1 peptideanalogs, whether or not conjugated to a half-life extending moiety, areuseful as immunosuppressive agents with therapeutic value for autoimmunediseases. For example, such molecules are useful in treating multiplesclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease, andrheumatoid arthritis. (See, e.g., H. Wulff et al. (2003) J. Clin.Invest. 111, 1703-1713 and H. Rus et al. (2005) PNAS 102, 11094-11099;Beeton et al., Targeting effector memory T cells with a selectiveinhibitor peptide of Kv1.3 channels for therapy of autoimmune diseases,Molec. Pharmacol. 67(4):1369-81 (2005); 1 Beeton et al. (2006), Kv1.3:therapeutic target for cell-mediated autoimmune disease, electronicpreprint at //webfiles.uci.edu/xythoswfs/webui/2670029.1). Inhibitors ofthe voltage-gated potassium channel Kv1.3 have been examined in avariety of preclinical animal models of inflammation. Small molecule andpeptide inhibitors of Kv1.3 have been shown to block delayed typehypersensitivity responses to ovalbumin [C. Beeton et al. (2005) Mol.Pharmacol. 67, 1369] and tetanus toxoid [G. C. Koo et al. (1999) Clin.Immunol. 197, 99]. In addition to suppressing inflammation in the skin,inhibitors also reduced antibody production [G. C. Koo et al. (1997) J.Immunol. 158, 5120]. Kv1.3 antagonists have shown efficacy in a ratadoptive-transfer experimental autoimmune encephalomyelitis (AT-EAE)model of multiple sclerosis (MS). The Kv1.3 channel is overexpressed onmyelin-specific T cells from MS patients, lending further support to theutility Kv1.3 inhibitors may provide in treating MS. Inflammatory boneresorption was also suppressed by Kv1.3 inhibitors in a preclinicaladoptive-transfer model of periodontal disease [P. Valverde et al.(2004) J. Bone Mineral Res. 19, 155]. In this study, inhibitorsadditionally blocked antibody production to a bacterial outer membraneprotein,—one component of the bacteria used to induce gingivalinflammation. Recently in preclinical rat models, efficacy of Kv1.3inhibitors was shown in treating pristane-induced arthritis and diabetes[C. Beeton et al. (2006) preprint available at//webfiles.uci.edu/xythoswfs/webui/_xy-2670029_(—)1.]. The Kv1.3 channelis expressed on all subsets of T cells and B cells, but effector memoryT cells and class-switched memory B cells are particularly dependent onKv1.3 [H. Wulff et al. (2004) J. Immunol. 173, 776].Gad5/insulin-specific T cells from patients with new onset type 1diabetes, myelin-specific T cells from MS patients and T cells from thesynovium of rheumatoid arthritis patients all overexpress Kv1.3 [C.Beeton et al. (2006) preprint at//webfiles.uci.edu/xythoswfs/webui/_xy-2670029_(—)1.]. Because micedeficient in Kv1.3 gained less weight when placed on a high fat diet [J.Xu et al. (2003) Human Mol. Genet. 12, 551] and showed altered glucoseutilization [J. Xu et al. (2004) Proc. Natl. Acad. Sci. 101, 3112],Kv1.3 is also being investigated for the treatment of obesity anddiabetes. Breast cancer specimens [M. Abdul et al. (2003) AnticancerRes. 23, 3347] and prostate cancer cell lines [S. P. Fraser et al.(2003) Pflugers Arch. 446, 559] have also been shown to express Kv1.3,and Kv1.3 blockade may be of utility for treatment of cancer. Disordersthat can be treated in accordance with the inventive method of treatingan autoimmune disorder, involving Kv1.3 inhibitor toxin peptide(s),include multiple sclerosis, type 1 diabetes, psoriasis, inflammatorybowel disease, contact-mediated dermatitis, rheumatoid arthritis,psoriatic arthritis, asthma, allergy, restinosis, systemic sclerosis,fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome,inflammatory bone resorption, transplant rejection, graft-versus-hostdisease, and systemic lupus erythematosus (SLE) and other forms oflupus.

Some of the cells that express the calcium-activated potassium ofintermediate conductance IKCa1 include T cells, B cells, mast cells andred blood cells (RBCs). T cells and RBCS from mice deficient in IKCa1show defects in volume regulation [T. Begenisich et al. (2004) J. Biol.Chem. 279, 47681]. Preclinical and clinical studies have demonstratedIKCa1 inhibitors utility in treating sickle cell anemia [J. W. Stockeret al. (2003) Blood 101, 2412]. Blockers of the IKCa1 channel have alsobeen shown to block EAE, indicating they may possess utility intreatment of MS [E. P. Reich et al. (2005) Eur. J. Immunol. 35, 1027].IgE-mediated histamine production from mast cells is also blocked byIKCa1 inhibitors [S. Mark Duffy et al. (2004) J. Allergy Clin. Immunol.114, 66], therefore they may also be of benefit in treating asthma. TheIKCa1 channel is overexpressed on activated T and B lymphocytes [H.Wulff et al. (2004) J. Immunol. 173, 776] and thus may show utility intreatment of a wide variety of immune disorders. Outside of the immunesystem, IKCa1 inhibitors have also shown efficacy in a rat model ofvascular restinosis and thus might represent a new therapeutic strategyto prevent restenosis after angioplasty [R. Kohler et al. (2003)Circulation 108, 1119]. It is also thought that IKCa1 antagonists are ofutility in treatment of tumor angiogenesis since inhibitors suppressedendothelial cell proliferation and angionenesis in vivo [I. Grgic et al.(2005) Arterioscler. Thromb. Vasc. Biol. 25, 704]. The IKCa1 channel isupregulated in pancreatic tumors and inhibitors blocked proliferation ofpancreatic tumor cell lines [H. Jager et al. (2004) Mol Pharmacol. 65,630]. IKCa1 antagonists may also represent an approach to attenuateacute brain damage caused by traumatic brain injury [F. Mauler (2004)Eur. J. Neurosci. 20, 1761]. Disorders that can be treated with IKCa1inhibitors include multiple sclerosis, asthma, psoriasis,contact-mediated dermatitis, rheumatoid & psoriatic arthritis,inflammatory bowel disease, transplant rejection, graft-versus-hostdisease, Lupus, restinosis, pancreatic cancer, tumor angiogenesis andtraumatic brain injury.

Accordingly, molecules of this invention incorporating peptideantagonists of the calcium-activated potassium channel of intermediateconductance, IKCa can be used to treat immune dysfunction, multiplesclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease,contact-mediated dermatitis, rheumatoid arthritis, psoriatic arthritis,asthma, allergy, restinosis, systemic sclerosis, fibrosis, scleroderma,glomerulonephritis, Sjogren syndrome, inflammatory bone resorption,transplant rejection, graft-versus-host disease, and lupus.

Accordingly, the present invention includes a method of treating anautoimmune disorder, which involves administering to a patient who hasbeen diagnosed with an autoimmune disorder, such as multiple sclerosis,type 1 diabetes, psoriasis, inflammatory bowel disease, contact-mediateddermatitis, rheumatoid arthritis, psoriatic arthritis, asthma, allergy,restinosis, systemic sclerosis, fibrosis, scleroderma,glomerulonephritis, Sjogren syndrome, inflammatory bone resorption,transplant rejection, graft-versus-host disease, or lupus, atherapeutically effective amount of the inventive composition of matter,whereby at least one symptom of the disorder is alleviated in thepatient. “Alleviated” means to be lessened, lightened, diminished,softened, mitigated (i.e., made more mild or gentle), quieted, assuaged,abated, relieved, nullified, or allayed, regardless of whether thesymptom of interest is entirely erased, eradicated, eliminated, orprevented in a particular patient.

The present invention is further directed to a method of preventing ormitigating a relapse of a symptom of multiple sclerosis, which methodinvolves administering to a patient, who has previously experienced atleast one symptom of multiple sclerosis, a prophylactically effectiveamount of the inventive composition of matter, such that the at leastone symptom of multiple sclerosis is prevented from recurring or ismitigated.

The inventive compositions of matter preferred for use in practicing theinventive method of treating an autoimmune disorder, e.g., inflammatorybowel disease (IBD, including Crohn's Disease and ulcerative colitis),and the method of preventing or mitigating a relapse of a symptom ofmultiple sclerosis include as P (conjugated as in Formula I), a Kv1.3 orIKCa1 antagonist peptide, such as a ShK peptide, an OSK1 peptide or anOSK1 peptide analog, a ChTx peptide and/or a Maurotoxin (MTx) peptide,or peptide analogs of any of these.

For example, the conjugated ShK peptide or ShK peptide analog cancomprise an amino acid sequence selected from the following:

-   -   SEQ ID NOS: 5, 88 through 200, 548 through 561, 884 through 950,        or 1295 through 1300 as set forth in Table 2.

The conjugated OSK1 peptide, or conjugated or unconjugated OSK1 peptideanalog, can comprise an amino acid sequence selected from the following:

-   -   SEQ ID NOS: 25, 294 through 298, 562 through 636, 980 through        1274, GVIINVSCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK (OSK1-S7) (SEQ ID        NO: 1303), or GVIINVSCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK        (OSK1-S7,K16,D20) (SEQ ID NO: 1308) as set forth in Table 7, or        any of SEQ ID NOS: 1391 through 4912, 4916, 4920 through 5006,        5009, 5010, and 5012 through 5015 as set forth in Table 7A,        Table 7B, Table 7C, Table 7D, Table 7E, Table 7F, Table 7G,        Table 7H, Table 7I, or Table 7J.

Also by way of example, a the conjugated MTX peptide, MTX peptideanalog, ChTx peptide or ChTx peptide analog can comprise an amino acidsequence selected from:

-   -   SEQ ID NOS: 20, 330 through 343, 1301, 1302, 1304 through 1307,        1309, 1311, 1312, or 1315 through 1336 as set forth in Table 13;        or SEQ ID NOS: 36, 59, 344 through 346, or 1369 through 1390 as        set forth in Table 14.

Also useful in these methods conjugated, or unconjugated, are a Kv1.3 orIKCa1 inhibitor toxin peptide analog that comprises an amino acidsequence selected from:

-   -   SEQ ID NOS: 88, 89, 92, 148 through 200, 548 through 561, 884        through 949, or 1295 through 1300 as set forth in Table 2; or    -   SEQ ID NOS: 980 through 1274,        GVIINVSCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK (OSK1-S7) (SEQ ID NO:        1303), or GVIINVSCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK        (OSK1-S7,K16,D20) (SEQ ID NO: 1308) as set forth in Table 7; or    -   SEQ ID NOS: 330 through 337, 341, 1301, 1302, 1304 through 1307,        1309, 1311, 1312, and 1315 through 1336 as set forth in Table        13.

In accordance with these inventive methods, a patient who has beendiagnosed with an autoimmune disorder, such as, but not limited tomultiple sclerosis, type 1 diabetes, psoriasis, inflammatory boweldisease, contact-mediated dermatitis, rheumatoid arthritis, psoraticarthritis, asthma, allergy, restinosis, systemic sclerosis, fibrosis,scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory boneresorption, transplant rejection, graft-versus-host disease, or lupus,or a patient who has previously experienced at least one symptom ofmultiple sclerosis, are well-recognizable and/or diagnosed by theskilled practitioner, such as a physician, familiar with autoimmunedisorders and their symptoms.

For example, symptoms of multiple sclerosis can include the following:

-   -   visual symptoms, such as, optic neuritis (blurred vision, eye        pain, loss of color vision, blindness); diplopia (double        vision); nystagmus (jerky eye movements); ocular dysmetria        (constant under- or overshooting eye movements); internuclear        opthalmoplegia (lack of coordination between the two eyes,        nystagmus, diplopia); movement and sound phosphenes (flashing        lights when moving eyes or in response to a sudden noise);        afferent pupillary defect (abnormal pupil responses);    -   motor symptoms, such as, paresis, monoparesis, paraparesis,        hemiparesis, quadraparesis (muscle weakness—partial or mild        paralysis); plegia, paraplegia, hemiplegia, tetraplegia,        quadraplegia (paralysis—total or near total loss of muscle        strength); spasticity (loss of muscle tone causing stiffness,        pain and restricting free movement of affected limbs);        dysarthria (slurred speech and related speech problems); muscle        atrophy (wasting of muscles due to lack of use); spasms, cramps        (involuntary contraction of muscles); hypotonia, clonus        (problems with posture); myoclonus, myokymia (jerking and        twitching muscles, tics); restless leg syndrome (involuntary leg        movements, especially bothersome at night); footdrop (foot drags        along floor during walking); dysfunctional reflexes (MSRs,        Babinski's, Hoffman's, Chaddock's);    -   sensory symptoms, such as, paraesthesia (partial numbness,        tingling, buzzing and vibration sensations); anaesthesia        (complete numbness/loss of sensation); neuralgia, neuropathic        and neurogenic pain (pain without apparent cause, burning,        itching and electrical shock sensations); L'Hermitte's (electric        shocks and buzzing sensations when moving head); proprioceptive        dysfunction (loss of awareness of location of body parts);        trigeminal neuralgia (facial pain);    -   coordination and balance symptoms, such as, ataxia (loss of        coordination); intention tremor (shaking when performing fine        movements); dysmetria (constant under- or overshooting limb        movements); vestibular ataxia (abnormal balance function in the        inner ear); vertigo (nausea/vomitting/sensitivity to travel        sickness from vestibular ataxia); speech ataxia (problems        coordinating speech, stuttering); dystonia (slow limb position        feedback); dysdiadochokinesia (loss of ability to produce        rapidly alternating movements, for example to move to a rhythm);    -   bowel, bladder and sexual symptoms, such as, frequent        micturation, bladder spasticity (urinary urgency and        incontinence); flaccid bladder, detrusor-sphincter dyssynergia        (urinary hesitancy and retention); erectile dysfunction (male        and female impotence); anorgasmy (inability to achieve orgasm);        retrograde ejaculation (ejaculating into the bladder); frigidity        (inability to become sexually aroused); constipation (infrequent        or irregular bowel movements); fecal urgency (bowel urgency);        fecal incontinence (bowel incontinence);    -   cognitive symptoms, such as, depression; cognitive dysfunction        (short-term and long-term memory problems, forgetfulness, slow        word recall); dementia; mood swings, emotional lability,        euphoria; bipolar syndrome; anxiety; aphasia, dysphasia        (impairments to speech comprehension and production); and    -   other symptoms, such as, fatigue; Uhthoff's Symptom (increase in        severity of symptoms with heat); gastroesophageal reflux (acid        reflux); impaired sense of taste and smell; epileptic seizures;        swallowing problems, respiratory problems; and sleeping        disorders.

By way of further example, symptoms of inflammatory bowel disease caninclude the following symptoms of Crohn's Disease or ulcerative colitis:

A. Symptoms of Crohn's disease can include:

-   -   Abdominal pain. The pain often is described as cramping and        intermittent, and the abdomen may be sore when touched.        Abdominal pain may turn to a dull, constant ache as the        condition progresses.    -   Diarrhea. Some patients may have diarrhea 10 to 20 times per        day. They may wake up at night and need to go to the bathroom.        Crohn's disease may cause blood in stools, but not always.    -   Loss of appetite.    -   Fever. In severe cases, fever or other symptoms that affect the        entire body may develop. A high fever may indicate a        complication involving infection, such as an abscess.    -   Weight loss. Ongoing symptoms, such as diarrhea, can lead to        weight loss. Too few red blood cells (anemia). Some patients        with Crohn's disease develop anemia because of low iron levels        caused by bloody stools or the intestinal inflammation itself.

B. The symptoms of ulcerative colitis may include:

-   -   Diarrhea or rectal urgency. Some patients may have diarrhea 10        to 20 times per day. The urge to defecate may wake patients at        night.    -   Rectal bleeding. Ulcerative colitis usually causes bloody        diarrhea and mucus. Patients also may have rectal pain and an        urgent need to empty the bowels.    -   Abdominal pain, often described as cramping. The patient's        abdomen may be sore when touched.    -   Constipation. This symptom may develop depending on what part of        the colon is affected.    -   Loss of appetite.    -   Fever. In severe cases, fever or other symptoms that affect the        entire body may develop.    -   Weight loss. Ongoing (chronic) symptoms, such as diarrhea, can        lead to weight loss.    -   Too few red blood cells (anemia). Some patients develop anemia        because of low iron levels caused by bloody stools or intestinal        inflammation.

The symptoms of multiple sclerosis and inflammatory bowel disease(including Crohn's Disease and ulcerative colitis) enumerated above, aremerely illustrative and are not intended to be an exhaustive descriptionof all possible symptoms experienced by a single patient or by severalsufferers in composite, and to which the present invention is directed.Those skilled in the art are aware of various clinical symptoms andconstellations of symptoms of autoimmune disorders suffered byindividual patients, and to those symptoms are also directed the presentinventive methods of treating an autoimmune disorder or of preventing ormitigating a relapse of a symptom of multiple sclerosis.

The therapeutically effective amount, prophylactically effective amount,and dosage regimen involved in the inventive methods of treating anautoimmune disorder or of preventing or mitigating a relapse of asymptom of multiple sclerosis, will be determined by the attendingphysician, considering various factors which modify the action oftherapeutic agents, such as the age, condition, body weight, sex anddiet of the patient, the severity of the condition being treated, timeof administration, and other clinical factors. Generally, the dailyamount or regimen should be in the range of about 1 to about 10,000micrograms (μg) of the vehicle-conjugated peptide per kilogram (kg) ofbody mass, preferably about 1 to about 5000 μg per kilogram of bodymass, and most preferably about 1 to about 1000 μg per kilogram of bodymass.

Molecules of this invention incorporating peptide antagonists of thevoltage-gated potassium channel Kv2.1 can be used to treat type IIdiabetes.

Molecules of this invention incorporating peptide antagonists of the Mcurrent (e.g., BeKm-1) can be used to treat Alzheimer's disease andenhance cognition.

Molecules of this invention incorporating peptide antagonists of thevoltage-gated potassium channel Kv4.3 can be used to treat Alzheimer'sdisease.

Molecules of this invention incorporating peptide antagonists of thecalcium-activated potassium channel of small conductance, SKCa can beused to treat epilepsy, memory, learning, neuropsychiatric,neurological, neuromuscular, and immunological disorders, schizophrenia,bipolar disorder, sleep apnea, neurodegeneration, and smooth muscledisorders.

Molecules of this invention incorporating N-type calcium channelantagonist peptides are useful in alleviating pain. Peptides with suchactivity (e.g., Ziconotide™, ω-conotoxin-MVIIA) have been clinicallyvalidated.

Molecules of this invention incorporating T-type calcium channelantagonist peptides are useful in alleviating pain. Several lines ofevidence have converged to indicate that inhibition of Cav3.2 in dorsalroot ganglia may bring relief from chronic pain. T-type calcium channelsare found at extremely high levels in the cell bodies of a subset ofneurons in the DRG; these are likely mechanoreceptors adapted to detectslowly-moving stimuli (Shin et al., Nature Neuroscience 6:724-730,2003), and T-type channel activity is likely responsible for burstspiking (Nelson et al., J Neurosci 25:8766-8775, 2005). Inhibition ofT-type channels by either mibefradil or ethosuximide reverses mechanicalallodynia in animals induced by nerve injury (Dogrul et al., Pain105:159-168, 2003) or by chemotherapy (Flatters and Bennett, Pain109:150-161, 2004). Antisense to Cav3.2, but not Cav3.1 or Cav3.3,increases pain thresholds in animals and also reduces expression ofCav3.2 protein in the DRG (Bourinet et al., EMBO J 24:315-324, 2005).Similarly, locally injected reducing agents produce pain and increaseCav3.2 currents, oxidizing agents reduce pain and inhibit Cav3.2currents, and peripherally administered neurosteroids are analgesic andinhibit T-type currents from DRG (Todorovic et al., Pain 109:328-339,2004; Pathirathna et al., Pain 114:429-443, 2005). Accordingly, it isthought that inhibition of Cav3.2 in the cell bodies of DRG neurons caninhibit the repetitive spiking of these neurons associated with chronicpain states.

Molecules of this invention incorporating L-type calcium channelantagonist peptides are useful in treating hypertension. Small moleculeswith such activity (e.g., DHP) have been clinically validated.

Molecules of this invention incorporating peptide antagonists of theNa_(V)1 (TTXs-type) channel can be used to alleviate pain. Localanesthetics and tricyclic antidepressants with such activity have beenclinically validated. Such molecules of this invention can in particularbe useful as muscle relaxants.

Molecules of this invention incorporating peptide antagonists of theNa_(V)1 (TTX_(R)-type) channel can be used to alleviate pain arisingfrom nerve and or tissue injury.

Molecules of this invention incorporating peptide antagonists of glial &epithelial cell Ca²⁺-activated chloride channel can be used to treatcancer and diabetes.

Molecules of this invention incorporating peptide antagonists of NMDAreceptors can be used to treat pain, epilepsy, brain and spinal cordinjury.

Molecules of this invention incorporating peptide antagonists ofnicotinic receptors can be used as muscle relaxants. Such molecules canbe used to treat pain, gastric motility disorders, urinary incontinence,nicotine addiction, and mood disorders.

Molecules of this invention incorporating peptide antagonists of 5HT3receptor can be used to treat Nausea, pain, and anxiety.

Molecules of this invention incorporating peptide antagonists of thenorepinephrine transporter can be used to treat pain, anti-depressant,learning, memory, and urinary incontinence.

Molecules of this invention incorporating peptide antagonists of theNeurotensin receptor can be used to treat pain.

In addition to therapeutic uses, the compounds of the present inventioncan be useful in diagnosing diseases characterized by dysfunction oftheir associated protein of interest. In one embodiment, a method ofdetecting in a biological sample a protein of interest (e.g., areceptor) that is capable of being activated comprising the steps of:(a) contacting the sample with a compound of this invention; and (b)detecting activation of the protein of interest by the compound. Thebiological samples include tissue specimens, intact cells, or extractsthereof. The compounds of this invention can be used as part of adiagnostic kit to detect the presence of their associated proteins ofinterest in a biological sample. Such kits employ the compounds of theinvention having an attached label to allow for detection. The compoundsare useful for identifying normal or abnormal proteins of interest.

The therapeutic methods, compositions and compounds of the presentinvention can also be employed, alone or in combination with othermolecules in the treatment of disease.

Pharmaceutical Compositions

In General. The present invention also provides pharmaceuticalcompositions comprising the inventive composition of matter and apharmaceutically acceptable carrier. Such pharmaceutical compositionscan be configured for administration to a patient by a wide variety ofdelivery routes, e.g., an intravascular delivery route such as byinjection or infusion, subcutaneous, intramuscular, intraperitoneal,epidural, or intrathecal delivery routes, or for oral, enteral,pulmonary (e.g., inhalant), intranasal, transmucosal (e.g., sublingualadministration), transdermal or other delivery routes and/or forms ofadministration known in the art. The inventive pharmaceuticalcompositions may be prepared in liquid form, or may be in dried powderform, such as lyophilized form. For oral or enteral use, thepharmaceutical compositions can be configured, for example, as tablets,troches, lozenges, aqueous or oily suspensions, dispersible powders orgranules, emulsions, hard or soft capsules, syrups, elixirs or enteralformulas.

In the practice of this invention the “pharmaceutically acceptablecarrier” is any physiologically tolerated substance known to those ofordinary skill in the art useful in formulating pharmaceuticalcompositions, including, any pharmaceutically acceptable diluents,excipients, dispersants, binders, fillers, glidants, anti-frictionalagents, compression aids, tablet-disintegrating agents (disintegrants),suspending agents, lubricants, flavorants, odorants, sweeteners,permeation or penetration enhancers, preservatives, surfactants,solubilizers, emulsifiers, thickeners, adjuvants, dyes, coatings,encapsulating material(s), and/or other additives singly or incombination. Such pharmaceutical compositions can include diluents ofvarious buffer content (e.g., Tris-HCl, acetate, phosphate), pH andionic strength; additives such as detergents and solubilizing agents(e.g., Tween® 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid,sodium metabisulfite), preservatives (e.g., Thimersol®, benzyl alcohol)and bulking substances (e.g., lactose, mannitol); incorporation of thematerial into particulate preparations of polymeric compounds such aspolylactic acid, polyglycolic acid, etc. or into liposomes. Hyaluronicacid can also be used, and this can have the effect of promotingsustained duration in the circulation. Such compositions can influencethe physical state, stability, rate of in vivo release, and rate of invivo clearance of the present proteins and derivatives. See, e.g.,Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack PublishingCo., Easton, Pa. 18042) pages 1435-1712, which are herein incorporatedby reference in their entirety. The compositions can be prepared inliquid form, or can be in dried powder, such as lyophilized form.Implantable sustained release formulations are also useful, as aretransdermal or transmucosal formulations. Additionally (oralternatively), the present invention provides compositions for use inany of the various slow or sustained release formulations ormicroparticle formulations known to the skilled artisan, for example,sustained release microparticle formulations, which can be administeredvia pulmonary, intranasal, or subcutaneous delivery routes.

One can dilute the inventive compositions or increase the volume of thepharmaceutical compositions of the invention with an inert material.Such diluents can include carbohydrates, especially, mannitol,α-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans andstarch. Certain inorganic salts may also be used as fillers, includingcalcium triphosphate, magnesium carbonate and sodium chloride. Somecommercially available diluents are Fast-Flo, Emdex, STA-Rx 1500,Emcompress and Avicell.

A variety of conventional thickeners are useful in creams, ointments,suppository and gel configurations of the pharmaceutical composition,such as, but not limited to, alginate, xanthan gum, or petrolatum, mayalso be employed in such configurations of the pharmaceuticalcomposition of the present invention. A permeation or penetrationenhancer, such as polyethylene glycol monolaurate, dimethyl sulfoxide,N-vinyl-2-pyrrolidone, N-(2-hydroxyethyl)-pyrrolidone, or3-hydroxy-N-methyl-2-pyrrolidone can also be employed. Useful techniquesfor producing hydrogel matrices are known. (E.g., Feijen, Biodegradablehydrogel matrices for the controlled release of pharmacologically activeagents, U.S. Pat. No. 4,925,677; Shah et al., BiodegradablepH/thermosensitive hydrogels for sustained delivery of biologicallyactive agents, WO 00/38651 A1). Such biodegradable gel matrices can beformed, for example, by crosslinking a proteinaceous component and apolysaccharide or mucopolysaccharide component, then loading with theinventive composition of matter to be delivered.

Liquid pharmaceutical compositions of the present invention that aresterile solutions or suspensions can be administered to a patient byinjection, for example, intramuscularly, intrathecally, epidurally,intravascularly (e.g., intravenously or intraarterially),intraperitoneally or subcutaneously. (See, e.g., Goldenberg et al.,Suspensions for the sustained release of proteins, U.S. Pat. No.6,245,740 and WO 00/38652 A1). Sterile solutions can also beadministered by intravenous infusion. The inventive composition can beincluded in a sterile solid pharmaceutical composition, such as alyophilized powder, which can be dissolved or suspended at a convenienttime before administration to a patient using sterile water, saline,buffered saline or other appropriate sterile injectable medium.

Implantable sustained release formulations are also useful embodimentsof the inventive pharmaceutical compositions. For example, thepharmaceutically acceptable carrier, being a biodegradable matriximplanted within the body or under the skin of a human or non-humanvertebrate, can be a hydrogel similar to those described above.Alternatively, it may be formed from a poly-alpha-amino acid component.(Sidman, Biodegradable, implantable drug delivery device, and processfor preparing and using same, U.S. Pat. No. 4,351,337). Other techniquesfor making implants for delivery of drugs are also known and useful inaccordance with the present invention.

In powder forms, the pharmaceutically acceptable carrier is a finelydivided solid, which is in admixture with finely divided activeingredient(s), including the inventive composition. For example, in someembodiments, a powder form is useful when the pharmaceutical compositionis configured as an inhalant. (See, e.g., Zeng et al., Method ofpreparing dry powder inhalation compositions, WO 2004/017918; Trunk etal., Salts of the CGRP antagonist BIBN4096 and inhalable powderedmedicaments containing them, U.S. Pat. No. 6,900,317).

One can dilute or increase the volume of the compound of the inventionwith an inert material. These diluents could include carbohydrates,especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose,modified dextrans and starch. Certain inorganic salts can also be usedas fillers including calcium triphosphate, magnesium carbonate andsodium chloride. Some commercially available diluents are Fast-Flo™,Emdex™, STA-Rx™ 1500, Emcompress™ and Avicell™.

Disintegrants can be included in the formulation of the pharmaceuticalcomposition into a solid dosage form. Materials used as disintegrantsinclude but are not limited to starch including the commercialdisintegrant based on starch, Explotab™. Sodium starch glycolate,Amberlite™, sodium carboxymethylcellulose, ultramylopectin, sodiumalginate, gelatin, orange peel, acid carboxymethyl cellulose, naturalsponge and bentonite can all be used. Insoluble cationic exchange resinis another form of disintegrant. Powdered gums can be used asdisintegrants and as binders and these can include powdered gums such asagar, Karaya or tragacanth. Alginic acid and its sodium salt are alsouseful as disintegrants.

Binders can be used to hold the therapeutic agent together to form ahard tablet and include materials from natural products such as acacia,tragacanth, starch and gelatin. Others include methyl cellulose (MC),ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic.

An antifrictional agent can be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants can be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantscan also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, Carbowax 4000and 6000.

Glidants that might improve the flow properties of the drug duringformulation and to aid rearrangement during compression might be added.The glidants can include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the compound of this invention into the aqueousenvironment a surfactant might be added as a wetting agent. Surfactantscan include anionic detergents such as sodium lauryl sulfate, dioctylsodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergentsmight be used and could include benzalkonium chloride or benzethoniumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the protein orderivative either alone or as a mixture in different ratios.

Oral dosage forms. Also useful are oral dosage forms of the inventivecompositions. If necessary, the composition can be chemically modifiedso that oral delivery is efficacious. Generally, the chemicalmodification contemplated is the attachment of at least one moiety tothe molecule itself, where said moiety permits (a) inhibition ofproteolysis; and (b) uptake into the blood stream from the stomach orintestine. Also desired is the increase in overall stability of thecompound and increase in circulation time in the body. Moieties usefulas covalently attached half-life extending moieties in this inventioncan also be used for this purpose. Examples of such moieties include:PEG, copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone andpolyproline. See, for example, Abuchowski and Davis (1981), SolublePolymer-Enzyme Adducts, Enzymes as Drugs (Hocenberg and Roberts, eds.),Wiley-Interscience, New York, N.Y., pp 367-83; Newmark, et al. (1982),J. Appl. Biochem. 4:185-9. Other polymers that could be used arepoly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred forpharmaceutical usage, as indicated above, are PEG moieties.

For oral delivery dosage forms, it is also possible to use a salt of amodified aliphatic amino acid, such as sodiumN-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), as a carrier to enhanceabsorption of the therapeutic compounds of this invention. The clinicalefficacy of a heparin formulation using SNAC has been demonstrated in aPhase II trial conducted by Emisphere Technologies. See U.S. Pat. No.5,792,451, “Oral drug delivery composition and methods.”

In one embodiment, the pharmaceutically acceptable carrier can be aliquid and the pharmaceutical composition is prepared in the form of asolution, suspension, emulsion, syrup, elixir or pressurizedcomposition. The active ingredient(s) (e.g., the inventive compositionof matter) can be dissolved, diluted or suspended in a pharmaceuticallyacceptable liquid carrier such as water, an organic solvent, a mixtureof both, or pharmaceutically acceptable oils or fats. The liquid carriercan contain other suitable pharmaceutical additives such as detergentsand/or solubilizers (e.g., Tween 80, Polysorbate 80), emulsifiers,buffers at appropriate pH (e.g., Tris-HCl, acetate, phosphate),adjuvants, anti-oxidants (e.g., ascorbic acid, sodium metabisulfite),preservatives (e.g., Thimersol, benzyl alcohol), sweeteners, flavoringagents, suspending agents, thickening agents, bulking substances (e.g.,lactose, mannitol), colors, viscosity regulators, stabilizers,electrolytes, osmolutes or osmo-regulators. Additives can also beincluded in the formulation to enhance uptake of the inventivecomposition. Additives potentially having this property are for instancethe fatty acids oleic acid, linoleic acid and linolenic acid.

Useful are oral solid dosage forms, which are described generally inRemington's Pharmaceutical Sciences (1990), supra, in Chapter 89, whichis hereby incorporated by reference in its entirety. Solid dosage formsinclude tablets, capsules, pills, troches or lozenges, cachets orpellets. Also, liposomal or proteinoid encapsulation can be used toformulate the present compositions (as, for example, proteinoidmicrospheres reported in U.S. Pat. No. 4,925,673). Liposomalencapsulation can be used and the liposomes can be derivatized withvarious polymers (e.g., U.S. Pat. No. 5,013,556). A description ofpossible solid dosage forms for the therapeutic is given in Marshall,K., Modern Pharmaceutics (1979), edited by G. S. Banker and C. T.Rhodes, in Chapter 10, which is hereby incorporated by reference in itsentirety. In general, the formulation will include the inventivecompound, and inert ingredients that allow for protection against thestomach environment, and release of the biologically active material inthe intestine.

The composition of this invention can be included in the formulation asfine multiparticulates in the form of granules or pellets of particlesize about 1 mm. The formulation of the material for capsuleadministration could also be as a powder, lightly compressed plugs oreven as tablets. The therapeutic could be prepared by compression.

Colorants and flavoring agents can all be included. For example, theprotein (or derivative) can be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

In tablet form, the active ingredient(s) are mixed with apharmaceutically acceptable carrier having the necessary compressionproperties in suitable proportions and compacted in the shape and sizedesired.

The powders and tablets preferably contain up to 99% of the activeingredient(s). Suitable solid carriers include, for example, calciumphosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch,gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ionexchange resins.

Controlled release formulation can be desirable. The composition of thisinvention could be incorporated into an inert matrix that permitsrelease by either diffusion or leaching mechanisms e.g., gums. Slowlydegenerating matrices can also be incorporated into the formulation,e.g., alginates, polysaccharides. Another form of a controlled releaseof the compositions of this invention is by a method based on the Oros™therapeutic system (Alza Corp.), i.e., the drug is enclosed in asemipermeable membrane which allows water to enter and push drug outthrough a single small opening due to osmotic effects. Some entericcoatings also have a delayed release effect.

Other coatings can be used for the formulation. These include a varietyof sugars that could be applied in a coating pan. The therapeutic agentcould also be given in a film-coated tablet and the materials used inthis instance are divided into 2 groups. The first are the nonentericmaterials and include methylcellulose, ethyl cellulose, hydroxyethylcellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl-methyl cellulose, sodium carboxymethyl cellulose,providone and the polyethylene glycols. The second group consists of theenteric materials that are commonly esters of phthalic acid.

A mix of materials might be used to provide the optimum film coating.Film coating can be carried out in a pan coater or in a fluidized bed orby compression coating.

Pulmonarv delivery forms. Pulmonary delivery of the inventivecompositions is also useful. The protein (or derivative) is delivered tothe lungs of a mammal while inhaling and traverses across the lungepithelial lining to the blood stream. (Other reports of this includeAdjei et al., Pharma. Res. (1990) 7: 565-9; Adjei et al. (1990),Internatl. J. Pharmaceutics 63: 135-44 (leuprolide acetate); Braquet etal. (1989), J. Cardiovasc. Pharmacol. 13 (supp1.5): s.143-146(endothelin-1); Hubbard et al. (1989), Annals Int. Med. 3: 206-12(α1-antitrypsin); Smith et al. (1989), J. Clin. Invest. 84: 1145-6(α1-proteinase); Oswein et al. (March 1990), “Aerosolization ofProteins,” Proc. Symp. Resp. Drug Delivery II, Keystone, Colo.(recombinant human growth hormone); Debs et al. (1988), J. Immunol. 140:3482-8 (interferon-γ and tumor necrosis factor α) and Platz et al., U.S.Pat. No. 5,284,656 (granulocyte colony stimulating factor).

Useful in the practice of this invention are a wide range of mechanicaldevices designed for pulmonary delivery of therapeutic products,including but not limited to nebulizers, metered dose inhalers, andpowder inhalers, all of which are familiar to those skilled in the art.Some specific examples of commercially available devices suitable forthe practice of this invention are the Ultravent nebulizer, manufacturedby Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer,manufactured by Marquest Medical Products, Englewood, Colo.; theVentolin metered dose inhaler, manufactured by Glaxo Inc., ResearchTriangle Park, N.C.; and the Spinhaler powder inhaler, manufactured byFisons Corp., Bedford, Mass. (See, e.g., Helgesson et al., Inhalationdevice, U.S. Pat. No. 6,892,728; McDerment et al., Dry powder inhaler,WO 02/11801 A1; Ohki et al., Inhalant medicator, U.S. Pat. No.6,273,086).

All such devices require the use of formulations suitable for thedispensing of the inventive compound. Typically, each formulation isspecific to the type of device employed and can involve the use of anappropriate propellant material, in addition to diluents, adjuvantsand/or carriers useful in therapy.

The inventive compound should most advantageously be prepared inparticulate form with an average particle size of less than 10 μm (ormicrons), most preferably 0.5 to 5 μm, for most effective delivery tothe distal lung.

Pharmaceutically acceptable carriers include carbohydrates such astrehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Otheringredients for use in formulations can include DPPC, DOPE, DSPC andDOPC. Natural or synthetic surfactants can be used. PEG can be used(even apart from its use in derivatizing the protein or analog).Dextrans, such as cyclodextran, can be used. Bile salts and otherrelated enhancers can be used. Cellulose and cellulose derivatives canbe used. Amino acids can be used, such as use in a buffer formulation.

Also, the use of liposomes, microcapsules or microspheres, inclusioncomplexes, or other types of carriers is contemplated.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise the inventive compound dissolved inwater at a concentration of about 0.1 to 25 mg of biologically activeprotein per mL of solution. The formulation can also include a bufferand a simple sugar (e.g., for protein stabilization and regulation ofosmotic pressure). The nebulizer formulation can also contain asurfactant, to reduce or prevent surface induced aggregation of theprotein caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the inventive compoundsuspended in a propellant with the aid of a surfactant. The propellantcan be any conventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid can also be useful as a surfactant. (See,e.g., Bäckström et al., Aerosol drug formulations containinghydrofluoroalkanes and alkyl saccharides, U.S. Pat. No. 6,932,962).

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing the inventive compound and can alsoinclude a bulking agent, such as lactose, sorbitol, sucrose, mannitol,trehalose, or xylitol in amounts which facilitate dispersal of thepowder from the device, e.g., 50 to 90% by weight of the formulation.

Nasal delivery forms. In accordance with the present invention,intranasal delivery of the inventive composition of matter and/orpharmaceutical compositions is also useful, which allows passage thereofto the blood stream directly after administration to the inside of thenose, without the necessity for deposition of the product in the lung.Formulations suitable for intransal administration include those withdextran or cyclodextran, and intranasal delivery devices are known.(See, e.g, Freezer, Inhaler, U.S. Pat. No. 4,083,368).

Transdermal and transmucosal (e.g., buccal) delivery forms). In someembodiments, the inventive composition is configured as a part of apharmaceutically acceptable transdermal or transmucosal patch or atroche. Transdermal patch drug delivery systems, for example, matrixtype transdermal patches, are known and useful for practicing someembodiments of the present pharmaceutical compositions. (E.g., Chien etal., Transdermal estrogen/progestin dosage unit, system and process,U.S. Pat. Nos. 4,906,169 and 5,023,084; Cleary et al., Diffusion matrixfor transdermal drug administration and transdermal drug deliverydevices including same, U.S. Pat. No. 4,911,916; Teillaud et al.,EVA-based transdermal matrix system for the administration of anestrogen and/or a progestogen, U.S. Pat. No. 5,605,702; Venkateshwaranet al., Transdermal drug delivery matrix for coadministering estradioland another steroid, U.S. Pat. No. 5,783,208; Ebert et al., Methods forproviding testosterone and optionally estrogen replacement therapy towomen, U.S. Pat. No. 5,460,820). A variety of pharmaceuticallyacceptable systems for transmucosal delivery of therapeutic agents arealso known in the art and are compatible with the practice of thepresent invention. (E.g., Heiber et al., Transmucosal delivery ofmacromolecular drugs, U.S. Pat. Nos. 5,346,701 and 5,516,523;Longenecker et al., Transmembrane formulations for drug administration,U.S. Pat. No. 4,994,439).

Buccal delivery of the inventive compositions is also useful. Buccaldelivery formulations are known in the art for use with peptides. Forexample, known tablet or patch systems configured for drug deliverythrough the oral mucosa (e.g., sublingual mucosa), include someembodiments that comprise an inner layer containing the drug, apermeation enhancer, such as a bile salt or fusidate, and a hydrophilicpolymer, such as hydroxypropyl cellulose, hydroxypropyl methylcellulose,hydroxyethyl cellulose, dextran, pectin, polyvinyl pyrrolidone, starch,gelatin, or any number of other polymers known to be useful for thispurpose. This inner layer can have one surface adapted to contact andadhere to the moist mucosal tissue of the oral cavity and can have anopposing surface adhering to an overlying non-adhesive inert layer.Optionally, such a transmucosal delivery system can be in the form of abilayer tablet, in which the inner layer also contains additionalbinding agents, flavoring agents, or fillers. Some useful systems employa non-ionic detergent along with a permeation enhancer. Transmucosaldelivery devices may be in free form, such as a cream, gel, or ointment,or may comprise a determinate form such as a tablet, patch or troche.For example, delivery of the inventive composition can be via atransmucosal delivery system comprising a laminated composite of, forexample, an adhesive layer, a backing layer, a permeable membranedefining a reservoir containing the inventive composition, a peel sealdisc underlying the membrane, one or more heat seals, and a removablerelease liner. (E.g., Ebert et al., Transdermal delivery system withadhesive overlay and peel seal disc, U.S. Pat. No. 5,662,925; Chang etal., Device for administering an active agent to the skin or mucosa,U.S. Pat. Nos. 4,849,224 and 4,983,395). These examples are merelyillustrative of available transmucosal drug delivery technology and arenot limiting of the present invention.

Dosages. The dosage regimen involved in a method for treating theabove-described conditions will be determined by the attendingphysician, considering various factors which modify the action of drugs,e.g. the age, condition, body weight, sex and diet of the patient, theseverity of any infection, time of administration and other clinicalfactors. Generally, the daily regimen should be in the range of 0.1-1000micrograms of the inventive compound per kilogram of body weight,preferably 0.1-150 micrograms per kilogram.

WORKING EXAMPLES

The compositions described above can be prepared as described below.These examples are not to be construed in any way as limiting the scopeof the present invention.

Example 1 Fc-L10-ShK[1-35] Mammalian Expression

Fc-L10-ShK[1-35], also referred to as “Fc-2xL-ShK[1-35]”, an inhibitorof Kv1.3. A DNA sequence coding for the Fc region of human IgG1 fusedin-frame to a linker sequence and a monomer of the Kv1.3 inhibitorpeptide ShK[1-35] was constructed as described below. Methods forexpressing and purifying the peptibody from mammalian cells (HEK 293 andChinese Hamster Ovary cells) are disclosed herein.

The expression vector pcDNA3.1(+) CMVi (FIG. 13A) was constructed byreplacing the CMV promoter between MluI and HindIII in pcDNA3.1(+) withthe CMV promoter plus intron (Invitrogen). The expression vectorpcDNA3.1 (+) CMVi-hFc-ActivinRIIB (FIG. 13B) was generated by cloning aHindIII-NotI digested PCR product containing a 5′ Kozak sequence, asignal peptide and the human Fc-linker-ActivinRIIB fusion protein withthe large fragment of HindIII-NotI digested pcDNA3.1(+) CMVi. Thenucleotide and amino acid sequence of the human IgG1 Fc region inpcDNA3.1(+) CMVi-hFc-ActivinRIIB is shown in FIG. 3A-3B. This vectoralso has a GGGGSGGGGS (“L10”; SEQ ID NO:79) linker split by a BamHI sitethus enabling with the oligo below formation of the 10 amino acid linkerbetween Fc and the ShK[1-35] peptide (see FIG. 14A-14B) for the finalFc-L10-ShK[1-35] nucleotide and amino acid sequence (FIG. 14A-14B andSEQ ID NO: 77 and SEQ ID NO:78).

The Fc-L10-ShK[1-35] expression vector was constructing using PCRstategies to generate the full length ShK gene linked to a four glycineand one serine amino acid linker (lower case letters here indicatelinker sequence of L-form amino acid residues) with two stop codons andflanked by BamHI and NotI restriction sites as shown below.

BamHIGGATCCGGAGGAGGAGGAAGCCGCAGCTGCATCGACACCATCCCCAAGAGCCGCTGCACCGCCTTCCAG//SEQ ID NO: 657      g  g  g  s  R  S  C  I  D  T  I  P  K  S  R  C  T  A  F  Q//SEQ ID NO: 658TGCAAGCACAGCATGAAGTACCGCCTGAGCTTCTGCCGCAAGACCTGCGGCACCTGCTAATGAGCGGCCGCC  K  H  S  M  K  Y  R  L  S  F  C  R  K  T  C  G  T  C          NotI

Two oligos with the sequence as depicted below were used in a PCRreaction with Herculase™ polymerase (Stratagene) at 94° C.-30 sec, 50°C.-30 sec, and 72° C.-1 min for 30 cycles.

//SEQ ID NO: 659 cat gga tcc gga gga gga gga agc cgc agc tgc atcgac acc atc ccc aag agc cgc tgc acc gcc ttc cag tgc aag cac//SEQ ID NO: 660 cat gcg gcc gct cat tag cag gtg ccg cag gtc ttgcgg cag aag ctc agg cgg tac ttc atg ctg tgc ttg cac tgg aag g

The resulting PCR products were resolved as the 150 bp bands on a onepercent agarose gel. The 150 bp PCR product was digested with BamHI andNotI (Roche) restriction enzymes and agarose gel purified by GelPurification Kit (Qiagen). At the same time, the pcDNA3.1 (+)CMVi-hFc-ActivinRIIB vector (FIG. 13B) was digested with BamHI and NotIrestriction enzymes and the large fragment was purified by GelPurification Kit. The gel purified PCR fragment was ligated to thepurified large fragment and transformed into XL-1 blue bacteria(Stratagene). DNAs from transformed bacterial colonies were isolated anddigested with BamHI and NotI restriction enzyme digestion and resolvedon a one percent agarose gel. DNAs resulting in an expected pattern weresubmitted for sequencing. Although, analysis of several sequences ofclones yielded a 100% percent match with the above sequence, only oneclone was selected for large scaled plasmid purification. The DNA fromFc-2xL-ShK in pcDNA3.1(+) CMVi clone was resequenced to confirm the Fcand linker regions and the sequence was 100% identical to the predictedcoding sequence, which is shown in FIG. 14A-14B.

HEK-293 cells used in transient transfection expression ofFc-2xL-ShK[1-35] in pcDNA3.1(+) CMVi protein were cultured in growthmedium containing DMEM High Glucose (Gibco), 10% fetal bovine serum (FBSfrom Gibco) and 1× non-essential amino acid (NEAA from Gibco). 5.6 ug ofFc-2xL-ShK[1-35] in pcDNA3.1(+) CMVi plasmid that had beenphenol/chloroform extracted was transfected into HEK-293 cells usingFugene 6 (Roche). The cells recovered for 24 hours, and then placed inDMEM High Glucose and 1× NEAA medium for 48 hours. The conditionedmedium was concentrated 50× by running 30 ml through Centriprep YM-10filter (Amicon) and further concentrated by a Centricon YM-10 (Amicon)filter. Various amounts of concentrated medium were mixed with anin-house 4× Loading Buffer (without B-mercaptoethanol) andelectrophoresed on a Novex 4-20% tris-glycine gel using a Novex Xcell IIapparatus at 101V/46 mA for 2 hours in a 5× Tank buffer solution (0.123Tris Base, 0.96M Glycine) along with 10 ul of BenchMark Pre-StainedProtein ladder (Invitrogen). The gel was then soaked in Electroblotbuffer (35 mM Tris base, 20% methanol, 192 mM glycine) for 30 minutes. APVDF membrane from Novex (Cat. No. LC2002, 0.2 um pores size) was soakedin methanol for 30 seconds to activate the PVDF, rinsed with deionizedwater, and soaked in Electroblot buffer. The pre-soaked gel was blottedto the PVDF membrane using the XCell II Blot module according to themanufacturer instructions (Novex) at 40 mA for 2 hours. Then, the blotwas first soaked in a 5% milk (Carnation) in Tris buffered salinesolution pH7.5 (TBS) for 1 hour at room temperature and incubated with1:500 dilution in TBS with 0.1% Tween-20 (TBST Sigma) and 1% milk bufferof the HRP-conjugated murine anti-human Fc antibody (Zymed LaboratoresCat. no. 05-3320) for two hours shaking at room temperature. The blotwas then washed three times in TBST for 15 minutes per wash at roomtemperature. The primary antibody was detected using Amersham PharmaciaBiotech's ECL western blotting detection reagents according tomanufacturer's instructions. Upon ECL detection, the western blotanalysis displayed the expected size of 66 kDa under non-reducing gelconditions (FIG. 24A).

AM1 CHOd- (Amgen Proprietary) cells used in the stable expression ofFc-L10-ShK[1-35] protein were cultured in AM1 CHOd- growth mediumcontaining DMEM High Glucose, 10% fetal bovine serum, 1×hypoxantine/thymidine (HT from Gibco) and 1×NEAA. 6.5 ug of pcDNA3.1(+)CMVi-Fc-ShK plasmid was also transfected into AM1 CHOd- cells usingFugene 6. The following day, the transfected cells were plated intotwenty 15 cm dishes and selected using DMEM high glucose, 10% FBS, 1xHT,1xNEAA and Geneticin (800 μg/ml G418 from Gibco) for thirteen days.Forty-eight surviving colonies were picked into two 24-well plates. Theplates were allowed to grow up for a week and then replicated forfreezing. One set of each plate was transferred to AM1 CHOd- growthmedium without 10% FBS for 48 hours and the conditioned media wereharvested. Western Blot analysis similar to the transient Western blotanalysis with detection by the same anti-human Fc antibody was used toscreen 15 ul of conditioned medium for expressing stable CHO clones. Ofthe 48 stable clones, more than 50% gave ShK expression at the expectedsize of 66 kDa. The BB6, BD5 and BD6 clones were selected with BD5 andBD6 as a backup to the primary clone BB6 (FIG. 24B).

The BB6 clone was scaled up into ten roller bottles (Corning) using AM1CHOd- growth medium and grown to confluency as judged under themicroscope. Then, the medium was exchanged with a serum-free mediumcontaining to 50% DMEM high glucose and 50% Ham's F12 (Gibco) with 1xHTand 1xNEAA and let incubate for one week. The conditioned medium washarvested at the one-week incubation time, filtered through 0.45 μmfilter (Corning) and frozen. Fresh serum-free medium was added andincubated for an additional week. The conditioned serum-free medium washarvested like the first time and frozen.

Purification of monovalent and bivalent dimeric Fc-L10-ShK(1-35).Approximately 4 L of conditioned medium was thawed in a water bath atroom temperature. The medium was concentrated to about 450 ml using aSatorius Sartocon Polysulfon 10 tangential flow ultra-filtrationcassette (0.1 m²) at room temperature. The retentate was then filteredthrough a 0.22 μm cellulose acetate filter with a pre-filter. Theretentate was then loaded on to a 5 ml Amersham HiTrap Protein A columnat 5 ml/min 7° C., and the column was washed with several column volumesof Dulbecco's phosphate buffered saline without divalent cations (PBS)and sample was eluted with a step to 100 mM glycine pH 3.0. The proteinA elution pool (approximately 9 ml) was diluted to 50 ml with water andloaded on to a 5 ml Amersham HiTrap SP-HP column in S-Buffer A (20 mMNaH₂PO₄, pH 7.0) at 5 ml/min and 7° C. The column was then washed withseveral column volumes S-Buffer A, and then developed using a lineargradient from 25% to 75% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.0) at5 ml/min followed by a step to 100% S-Buffer B at 7° C. Fractions werethen analyzed using a Coomassie brilliant blue stained tris-glycine4-20% SDS-PAGE, and the fractions containing the desired product werepooled based on these data. The pooled material was then concentrated toabout 3.4 ml using a Pall Life Sciences Macrosep 10K Omega centrifugalultra-filtration device and then filtered though a Costar 0.22 μmcellulose acetate syringe filter.

A spectral scan was then conducted on 10 μl of the filtered materialdiluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer(FIG. 26A). The concentration of the filtered material was determined tobe 5.4 mg/ml using a calculated molecular mass of 32,420 g/mol andextinction coefficient of 47,900 M⁻¹ cm⁻¹.

The purity of the filtered bivalent dimeric Fc-L10-ShK(1-35) product wasassessed using a Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE (FIG. 26B). The monvalent dimeric Fc-L10-ShK(1-35) product wasanalyzed using reducing and non-reducing sample buffers by SDS-PAGE on a1.0 mm TRIS-glycine 4-20% gel developed at 220 V and stained with BostonBiologicals QuickBlue (FIG. 26E). The endotoxin levels were thendetermined using a Charles River Laboratories Endosafe-PTS system(0.05-5 EU/ml sensitivity) using a 108-fold dilution of the sample inPBS yielding a result of <1 EU/mg protein. The macromolecular state ofthe products was then determined using size exclusion chromatography on20 μg of the product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm (FIG. 26C, bivalent dimeric Fc-L10-ShK(1-35);FIG. 26F, monovalent dimeric Fc-L10-SHK(1-35)). The product was thensubject to mass spectral analysis by diluting 1 μl of the sample into 10μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50%acetonitrile). The resultant solution (1 μl) was spotted onto a MALDIsample plate. The sample was allowed to dry before being analyzed usinga Voyager DE-RP time-of-flight mass spectrometer equipped with anitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode wasused, with an accelerating voltage of 25 kV. Each spectrum was producedby accumulating data from ˜200 laser shots. External mass calibrationwas accomplished using purified proteins of known molecular masses (FIG.26D) and confirmed (within experimental error) the integrity of thepurified peptibody. The product was then stored at −80° C.

Purified bivalent dimeric Fc-L10-ShK[1-35] potently blocked human Kv1.3(FIG. 30A and FIG. 30B) as determined by electrophysiology (see Example36). The purified bivalent dimeric Fc-L10-ShK[1-35] molecule alsoblocked T cell proliferation (FIG. 36A and FIG. 36B) and production ofthe cytokines IL-2 (FIG. 35A and FIG. 37A) and IFN-g (FIG. 35B and FIG.37B).

Example 2 Fc-L-ShK[2-35] Mammalian Expression

A DNA sequence coding for the Fc region of human IgG1 fused in-frame toa monomer of the Kv1.3 inhibitor peptide ShK[2-35] was constructed usingstandard PCR technology. The ShK[2-35] and the 5, 10, or 25 amino acidlinker portion of the molecule were generated in a PCR reaction usingthe original Fc-2xL-ShK[1-35] in pcDNA3.1(+) CMVi as a template (Example1, FIG. 14A-14B). All ShK constructs should have the following aminoacid sequence of

SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC (SEQ ID NO: 92)with the first amino acid of the wild-type sequence deleted.

The sequences of the primers used to generate Fc-L5-ShK[2-35], alsoreferred to as “Fc-1xL-ShK[2-35]”, are shown below:

//SEQ ID NO: 661 cat gga tcc agc tgc atc gac acc atc; //SEQ ID NO: 662cat gcg gcc gct cat tag c;

The sequences of the primers used to generate Fc-L10-ShK[2-35], alsoreferred to as “Fc-2xL-ShK[2-35]” are shown below:

//SEQ ID NO: 663 cat gga tcc gga gga gga gga agc agc tgc a;//SEQ ID NO: 664 cat gcg gcc gct cat tag cag gtg c;

The sequences of the primers used to generate Fc-L25-ShK[2-35], alsoreferred to as “Fc-5xL-ShK[2-35]”, are shown below:

//SEQ ID NO: 665 cat gga tcc ggg ggt ggg ggt tct ggg ggt ggg ggttct gga gga gga gga agc gga gga gga gga agc agc tgc a; //SEQ ID NO: 666cat gcg gcc gct cat tag cag gtg c;

The PCR products were digested with BamHI and NotI (Roche) restrictionenzymes and agarose gel purified by Gel Purification Kit. At the sametime, the pcDNA3.1(+) CMVi-hFc-ActivinRIIB vector was digested withBamHI and NotI restriction enzymes and the large fragment was purifiedby Gel Purification Kit. Each purified PCR product was ligated to thelarge fragment and transformed into XL-1 blue bacteria. DNAs fromtransformed bacterial colonies were isolated and subjected to BamHI andNotI restriction enzyme digestions and resolved on a one percent agarosegel. DNAs resulting in an expected pattern were submitted forsequencing. Although, analysis of several sequences of clones yielded a100% percent match with the above sequence, only one clone was selectedfor large scaled plasmid purification. The DNA from this clone wasresequenced to confirm the Fc and linker regions and the sequence was100% identical to the expected sequence.

Plasmids containing the Fc-1xL-Shk[2-35], Fc-2xL-Shk[2-35] andFc-5xL-Shk[2-35] inserts in pcDNA3.1 (+) CMVi vector were digested withXba1 and Xho1 (Roche) restriction enzymes and gel purified. The insertswere individually ligated into Not1 and SalI (Roche) digested pDSRα-22(Amgen Proprietary) expression vector. Integrity of the resultingconstructs were confirmed by DNA sequencing. The final plasmid DNAexpression vector constructs were pDSRα-22-Fc-1xL-Shk[2-35],pDSRα-22-Fc-2xL-Shk[2-35] (FIG. 13C and FIG. 15A-15B) andpDSRα-22-Fc-5xL-Shk[2-35] (FIG. 16A-16B) and contained 5, 10 and 25amino acid linkers, respectively.

Twenty-four hours prior to transfection, 1.2e7 AM-1/D CHOd- (AmgenProprietary) cells were plated into a T-175 cm sterile tissue cultureflask, to allow 70-80% confluency on the day of transfection. The cellshad been maintained in the AM-1/D CHOd- culture medium containing DMEMHigh Glucose, 5% FBS, 1× Glutamine Pen/Strep (Gibco), 1×HT, 1×NEAA's and1× sodium pyruvate (Gibco). The following day, eighteen micrograms ofeach of the linearized pDSRα22:Fc-1xL-ShK[2-35],pDSRα22:Fc-2xL-ShK[2-35] and pDSRα22:Fc-5xL-ShK[2-35] (RDS's#20050037685, 20050053709, 20050073295) plasmids were mixed with 72 μgof linearized Selexis MAR plasmid and pPAGO1 (RDS 20042009896) anddiluted into 6 ml of OptiMEM in a 50 ml conical tube and incubate forfive minutes. LF2000 (210 μl) was added to 6 ml of OptiMEM and incubatedfor five minutes. The diluted DNA and LF2000 were mixed together andincubated for 20 minutes at room temperature. In the meantime, the cellswere washed one time with PBS and then 30 ml OptiMEM without antibioticswere added to the cells. The OptiMEM was aspirated off, and the cellswere incubated with 12 ml of DNA/LF2000 mixture for 6 hours or overnightin the 37° C. incubator with shaking. Twenty-four hours posttransfection, the cells were split 1:5 into AM-1/D CHOd- culture mediumand at differing dilutions for colony selection. Seventy-two hours posttransfection, the cell medium was replaced with DHFR selection mediumcontaining 10% Dialyzed FBS (Gibco) in DMEM High Glucose, plus 1×Glutamine Pen/Strep, 1×NEAA's and 1× Na Pyr to allow expression andsecretion of protein into the cell medium. The selection medium waschanged two times a week until the colonies are big enough to pick. ThepDSRa22 expression vector contains a DHFR expression cassette, whichallows transfected cells to grow in the absence of hypoxanthine andthymidine. The five T-175 pools of the resulting colonies were scaled upinto roller bottles and cultured under serum free conditions. Theconditioned media were harvested and replaced at one-week intervals. Theresulting 3 liters of conditioned medium was filtered through a 0.45 μmcellulose acetate filter (Corning, Acton, Mass.) and transferred toProtein Chemistry for purification. As a backup, twelve colonies wereselected from the 10 cm plates after 10-14 days on DHFR selection mediumand expression levels evaluated by western blot using HRP conjugatedanti human IgGFc as a probe. The three best clones expressing thehighest level of each of the different linker length Fc-L-ShK[2-35]fusion proteins were expanded and frozen for future use.

Purification of Fc-L10-ShK(2-35). Approximately 1 L of conditionedmedium was thawed in a water bath at room temperature. The medium wasloaded on to a 5 ml Amersham HiTrap Protein A column at 5 ml/min 7° C.,and the column was washed with several column volumes of Dulbecco'sphosphate buffered saline without divalent cations (PBS) and sample waseluted with a step to 100 mM glycine pH 3.0. The protein A elution pool(approximately 8.5 ml) combined with 71 μl 3 M sodium acetate and thendiluted to 50 ml with water. The diluted material was then loaded on toa 5 ml Amersham HiTrap SP-HP column in S-Buffer A (20 mM NaH₂PO₄, pH7.0) at 5 ml/min 7° C. The column was then washed with several columnvolumes S-Buffer A, and then developed using a linear gradient from 0%to 75% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.0) at 5 ml/min followedby a step to 100% S-Buffer B at 7° C. Fractions were then analyzed usinga Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and thefractions containing the desired product were pooled based on thesedata. The pooled material was then filtered through a 0.22 μm celluloseacetate filter and concentrated to about 3.9 ml using a Pall LifeSciences Macrosep 10K Omega centrifugal ultra-filtration device. Theconcentrated material was then filtered though a Pall Life SciencesAcrodisc with a 0.22 μm, 25 mm Mustang E membrane at 2 ml/min roomtemperature. A spectral scan was then conducted on 10 μl of the filteredmaterial diluted in 700 μl PBS using a Hewlett Packard 8453spectrophotometer (FIG. 27E). The concentration of the filtered materialwas determined to be 2.76 mg/ml using a calculated molecular mass of30,008 g/mol and extinction coefficient of 36,900 M⁻¹ cm⁻¹. Sincematerial was found in the permeate, repeated concentration step on thepermeate using a new Macrosep cartridge. The new batch of concentratedmaterial was then filtered though a Pall Life Sciences Acrodisc with a0.22 μm, 25 mm Mustang E membrane at 2 ml/min room temperature. Bothlots of concentrated material were combined into one pool.

A spectral scan was then conducted on 10 μl of the combined pool dilutedin 700 μl PBS using a Hewlett Packard 8453 spectrophotometer. Theconcentration of the filtered material was determined to be 3.33 mg/mlusing a calculated molecular mass of 30,008 g/mol and extinctioncoefficient of 36,900 M⁻¹ cm⁻¹. The purity of the filtered material wasthen assessed using a Coomassie brilliant blue stained tris-glycine4-20% SDS-PAGE (FIG. 27A). The endotoxin level was then determined usinga Charles River Laboratories Endosafe-PTS system (0.05-5 EU/mlsensitivity) using a 67-fold dilution of the sample in PBS yielding aresult of <1 EU/mg protein. The macromolecular state of the product wasthen determined using size exclusion chromatography on 50 μg of theproduct injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm)in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing theabsorbance at 280 nm (FIG. 27B). The product was then subject to massspectral analysis by diluting 1 μl of the sample into 10 μl of sinapinicacid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). Theresultant solution (1 μl) was spotted onto a MALDI sample plate. Thesample was allowed to dry before being analyzed using a Voyager DE-RPtime-of-flight mass spectrometer equipped with a nitrogen laser (337 nm,3 ns pulse). The positive ion/linear mode was used, with an acceleratingvoltage of 25 kV. Each spectrum was produced by accumulating data from˜200 laser shots. External mass calibration was accomplished usingpurified proteins of known molecular masses (FIG. 27F) and theexperiment confirmed the integrity of the peptibody, within experimentalerror. The product was then stored at −80° C.

FIG. 31B shows that purified Fc-L10-ShK[2-35] potently blocks humanKv1.3 current (electrophysiology was done as described in Example 36).The purified Fc-L10-ShK[2-35] molecule also blocked IL-2 (FIG. 64A andFIG. 64B) and IFN-g (FIG. 65A and FIG. 65B) production in human wholeblood, as well as, upregulation of CD40L (FIG. 66A and FIG. 66B) andIL-2R (FIG. 67A and FIG. 67B) on T cells.

Purification of Fc-L5-ShK(2-35). Approximately 1 L of conditioned mediumwas loaded on to a 5 ml Amersham HiTrap Protein A column at 5 ml/min 7°C., and the column was washed with several column volumes of Dulbecco'sphosphate buffered saline without divalent cations (PBS) and sample waseluted with a step to 100 mM glycine pH 3.0. The protein A elution pool(approximately 9 ml) combined with 450 μl 1 M tris HCl pH 8.5 followedby 230 μl 2 M acetic acid then diluted to 50 ml with water. The pHadjusted material was then filtered through a 0.22 μm cellulose acetatefilter and loaded on to a 5 ml Amersham HiTrap SP-HP column in S-BufferA (20 mM NaH₂PO₄, pH 7.0) at 5 ml/min 7° C. The column was then washedwith several column volumes S-Buffer A, and then developed using alinear gradient from 0% to 75% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH7.0) at 5 ml/min followed by a step to 100% S-Buffer B at 7° C.Fractions were then analyzed using a Coomassie brilliant blue stainedtris-glycine 4-20% SDS-PAGE, and the fractions containing the desiredproduct were pooled based on these data. The pooled material was thenconcentrated to about 5.5 ml using a Pall Life Sciences Macrosep 10KOmega centrifugal ultra-filtration device. The concentrated material wasthen filtered though a Pall Life Sciences Acrodisc with a 0.22 μm, 25 mmMustang E membrane at 2 ml/min room temperature.

A spectral scan was then conducted on 10 μl of the combined pool dilutedin 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 27G).The concentration of the filtered material was determined to be 4.59mg/ml using a calculated molecular mass of 29,750 g/mol and extinctioncoefficient of 36,900 M⁻¹ cm⁻¹. The purity of the filtered material wasthen assessed using a Coomassie brilliant blue stained tris-glycine4-20% SDS-PAGE (FIG. 27C). The endotoxin level was then determined usinga Charles River Laboratories Endosafe-PTS system (0.05-5 EU/mlsensitivity) using a 92-fold dilution of the sample in Charles RiversEndotoxin Specific Buffer BG120 yielding a result of <1 EU/mg protein.The macromolecular state of the product was then determined using sizeexclusion chromatography on 50 μg of the product injected on to aPhenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mMNaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 27H).The product was then subject to mass spectral analysis by diluting 1 μlof the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05%trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl)was spotted onto a MALDI sample plate. The sample was allowed to drybefore being analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses (FIG. 27I) and confirmed theintegrity of the peptibody, within experimental error. The product wasthen stored at −80° C.

FIG. 31C shows that purified Fc-L5-ShK[2-35] is highly active and blockshuman Kv1.3 as determined by whole cell patch clamp electrophysiology(see Example 36).

Purification of Fc-L25-ShK(2-35). Approximately 1 L of conditionedmedium was loaded on to a 5 ml Amersham HiTrap Protein A column at 5ml/min 7° C., and the column was washed with several column volumes ofDulbecco's phosphate buffered saline without divalent cations (PBS) andsample was eluted with a step to 100 mM glycine pH 3.0. The protein Aelution pool (approximately 9.5 ml) combined with 119 μl 3 M sodiumacetate and then diluted to 50 ml with water. The pH adjusted materialwas then loaded on to a 5 ml Amersham HiTrap SP-HP column in S-Buffer A(20 mM NaH₂PO₄, pH 7.0) at 5 ml/min 7° C. The column was then washedwith several column volumes S-Buffer A, and then developed using alinear gradient from 0% to 75% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH7.0) at 5 ml/min followed by a step to 100% S-Buffer B at 7° C.Fractions containing the main peak from the chromatogram were pooled andfiltered through a 0.22 μm cellulose acetate filter.

A spectral scan was then conducted on 20 μl of the combined pool dilutedin 700 μl PBS using a Hewlett Packard 8453 spectrophotometer FIG. 27J.The concentration of the filtered material was determined to be 1.40mg/ml using a calculated molecular mass of 31,011 g/mol and extinctioncoefficient of 36,900 M⁻¹ cm⁻¹. The purity of the filtered material wasthen assessed using a Coomassie brilliant blue stained tris-glycine4-20% SDS-PAGE (FIG. 27D). The endotoxin level was then determined usinga Charles River Laboratories Endosafe-PTS system (0.05-5 EU/mlsensitivity) using a 28-fold dilution of the sample in Charles RiversEndotoxin Specific Buffer BG120 yielding a result of <1 EU/mg protein.The macromolecular state of the product was then determined using sizeexclusion chromatography on 50 μg of the product injected on to aPhenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mMNaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 27K).The product was then subject to mass spectral analysis by diluting 1 μlof the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05%trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl)was spotted onto a MALDI sample plate. The sample was allowed to drybefore being analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses (FIG. 27L) and this confirmed theintegrity of the peptibody, within experimental error. The product wasthen stored at −80° C.

Purified Fc-L25-ShK[2-35] inhibited human Kv1.3 with an IC₅₀ of ˜150 pMby whole cell patch clamp electrophysiology on HEK293/Kv1.3 cells(Example 36).

Example 3 Fc-L-ShK[1-35] Bacterial Expression

Description of bacterial peptibody expression vectors and procedures forcloning and expression of peptibodies. The cloning vector used forbacterial expression (Examples 3-30) is based on pAMG21 (originallydescribed in U.S. Patent 2004/0044188). It has been modified in that thekanamycin resistance component has been replaced with ampicillinresistance by excising the DNA between the unique BstBI and NsiI sitesof the vector and replacing with an appropriately digested PCR fragmentbearing the beta-lactamase gene using PCR primers CCA ACA CAC TTC GAAAGA CGT TGA TCG GCA C (SEQ ID NO: 667) and CAC CCA ACA ATG CAT CCT TAAAAA AAT TAC GCC C (SEQ ID NO: 668) with pUC19 DNA as the template sourceof the beta-lactamase gene conferring resistance to ampicillin. The newversion is called pAMG21ampR.

Description of cloning vector pAMG21ampR-Fc-Pep used in examples 3 to30, excluding 15 and 16. FIG. 11A-C and FIG. 11D (schematic diagram)show the ds-DNA that has been added to the basic vector pAMG21ampR topermit the cloning of peptide fusions to the C-terminus of the Fc gene.The DNA has been introduced between the unique NdeI and BamHI sites inthe pAMG21ampR vector. This entire region of DNA is shown in FIG. 11A-C.The coding region for Fc extends from nt 5134 to 5817 and the proteinsequence appears below the DNA sequence. This is followed in frame by aglyX5 linker (nt's 5818-5832). A BsmBI site (GAGACG) spansnucleotides5834-5839. DNA cleavage occurs between nucleotides 5828 and 5829 on theupper DNA strand and between nucleotides 5832 and 5833 on the lower DNAstrand. Digestion creates 4 bp cohesive termini as shown here. The BsmBIsite is underlined.

AGGTGG TGGTTGAGACG  SEQ ID NO: 683 TCCACCACCA     ACTCTGC SEQ ID NO: 684

A second BsmBI site occurs at nucleotides 6643 through 6648; viz.,CGTCTC. DNA cleavage occurs between nucleotides 6650 and 6651 on theupper strand and between 6654 and 6655 on the lower strand.

CGTCTCT TAAGGATCCG  SEQ ID NO: 685 GCAGAGAATTC     CTAGGC SEQ ID NO: 686

Between the two BsmBI sites is a dispensable chloramphenicol resistancecassette constitutively expressing chloramphenicol acetyltransferase(cat gene). The cat protein sequence:

1 MEKKITGYTT VDISQWHRKE HFEAFQSVAQ CTYNQTVQLD ITAFLKTVKK SEQ ID NO: 133751 NKHKFYPAFI HILARLMNAH PEFRMAMKDG ELVIWDSVHP CYTVFHEQTE 101TFSSLWSEYH DDFRQFLHIY SQDVACYGEN LAYFPKGFIE NMFFVSANPW 151VSFTSFDLNV ANMDNFFAPV FTMGKYYTQG DKVLMPLAIQ VHHAVCDGFH 201VGRMLNELQQ YCDEWQGGA//is shown in FIG. 11A-C and extends from nucleotides 5954 to 6610. Thepeptide encoding duplexes in each example (except Examples 15 and 16)bear cohesive ends complementary to those presented by the vector.

Description of the cloning vector pAMG21ampR-Pep-Fc used in examples 15and 16. FIG. 12A-C, and the schematic diagram in FIG. 12D, shows theds-DNA sequence that has been added to the basic vector pAMG21ampR topermit the cloning of peptide fusions to the N-terminus of the Fc gene.The DNA has been introduced between the unique NdeI and BamHI sites inthe pAMG21ampR vector. The coding region for Fc extends from nt 5640 to6309 and the protein sequence appears below the DNA sequence. This ispreceded in frame by a glyX5 linker (nt's 5614-5628). A BsmBI site spansnucleotides 5138 to 5143; viz., GAGACG. The cutting occurs betweennucleotides 5132 and 5133 on the upper DNA strand and between 5136 and5137 on the lower DNA strand.

Digestion creates 4 bp cohesive termini as shown. The BsmBI site isunderlined.

AATAACA TATGCGAGACG SEQ ID NO: 687 TTATTGTATAC     GCTCTGCSEQ ID NO: 688A second BsmBI site occurs at nucleotides 5607 through 5612; viz.,CGTCTC. Cutting occurs between nucleotides 5613 and 5614 on the upperstrand and between 5617 and 5618 on the lower strand.

CGTCTCA GGTGGTGGT SEQ ID NO: 689 GCAGAGTCCAC     CACCABetween the BsmBI sites is a dispensable zeocin resistance cassetteconstitutively expressing the Shigella ble protein. The ble proteinsequence:

1 MAKLTSAVPV LTARDVAGAV EFWTDRLGFS RDFVEDDFAG VVRDDVTLFI//SEQ ID NO: 1338 51SAVQDQVVPD NTLAWVWVRG LDELYAEWSE VVSTNFRDAS GPAMTEIGEQ 101PWGREFALRD PAGNCVHFVA EEQDis shown extending from nucleotides 5217 to 5588 in FIG. 12A-C. Thepeptide encoding duplexes in Examples 15 and 16 bear cohesive endscomplementary to those presented by the vector.

Description of the cloning vector pAMG21ampR-Pep-Fc used in Examples 52and 53. FIG. 12E-G shows the ds-DNA sequence that has been added to thebasic vector pAMG21ampR to permit the cloning of peptide fusions to theN-terminus of the Fc gene in which the first two codons of the peptideare to be met-gly. The DNA has been introduced between the unique NdeIand BamHI sites in the pAMG21ampR vector. The coding region for Fcextends from nt 5632 to 6312 and the protein sequence appears below theDNA sequence. This is preceded in frame by a glyX5 linker (nt's5617-5631). A BsmBI site spans nucleotides 5141 to 5146; viz., GAGACG.The cutting occurs between nucleotides 5135 and 5136 on the upper DNAstrand and between 5139 and 5140 on the lower DNA strand.

Digestion creates 4 bp cohesive termini as shown. The BsmBI site isunderlined.

AATAACATAT GGGTCGAGACG  SEQ ID NO: 1344 SEQ ID NO: 1343 TTATTGTATACCCA    GCTCTGC SEQ ID NO: 1345A second BsmBI site occurs at nucleotides 5607 through 5612; viz.,CGTCTC. Cutting occurs between nucleotides 5613 and 5614 on the upperstrand and between 5617 and 5618 on the lower strand.

CGTCTCA     GGTGGTGGT SEQ ID NO: 1346 GCAGAGTCCAC     CACCABetween the BsmBI sites is a dispensable zeocin resistance cassetteconstitutively expressing the Shigella ble protein. The ble proteinsequence, as described above, is shown extending from nucleotidepositions 5220 to 5591. The peptide encoding duplexes in Examples 52 and53 herein below bear cohesive ends complementary to those presented bythe vector.

For Examples 3 to 30 for which all are for bacterial expression, clonedpeptide sequences are all derived from the annealing of oligonucleotidesto create a DNA duplex that is directly ligated into the appropriatevector. Two oligos suffice for Example 20, four are required for allother examples. When the duplex is to be inserted at the N-terminus ofFc (see, Examples 15, 16, 52, and 53 herein) the design is as followswith the ordinal numbers matching the listing of oligos in each example:

When the duplex is to be inserted at the C-terminus of Fc (Examples 3,4, 5, 10, 11, 12, 13, and 30) the design is as follows:

All remaining examples have the duplex inserted at the C-terminus of Fcand utilize the following design.

No kinasing step is required for the phosphorylation of any of theoligos. A successful insertion of a duplex results in the replacement ofthe dispensable antibiotic resistance cassette (Zeocin resistance forpAMG21ampR-Pep-Fc and chloramphenicol resistance for pAMG21ampR-Fc-Pep).The resulting change in phenotype is useful for discriminatingrecombinant from nonrecombinant clones.

The following description gives the uniform method for carrying out thecloning of all 30 bacterially expressed recombinant proteins exemplifiedherein. Only the set of oligonucleotides and the vector are varied.These specifications are given below in each example.

An oligonucleotide duplex containing the coding region for a givenpeptide was formed by annealing the oligonucleotides listed in eachexample. Ten picomoles of each oligo was mixed in a final volume of 10μl containing 1× ligation buffer along with 0.3 μg of appropriate vectorthat had been previously digested with restriction endonuclease BsmBI.The mix was heated to 80° C. and allowed to cool at 0.1 degree/sec toroom temperature. To this was added 10 μl of 1× ligase buffer plus 400units of T4 DNA ligase. The sample was incubated at 14 C for 20 min.Ligase was inactivated by heating at 65° C. for 10 minutes. Next, 10units of restriction endonucleases BsmBI were added followed byincubation at 55 C for one hour to cleave any reformed parental vectormolecules. Fifty ul of chemically competent E. coli cells were added andheld at 2 C for 20 minutes followed by heat shock at 42 C for 5 second.The entire volume was spread onto Luria Agar plates supplemented withcarbenicillin at 200 μg/ml and incubated overnight at 37 C. Colonieswere tested for the loss of resistance to the replaceable antibioticresistance marker. A standard PCR test can be used to confirm theexpected size of the duplex insert. Plasmid preparations were obtainedand the recombinant insert was verified by DNA sequencing. Half litercultures of a sequence confirmed construct were grown in Terrific Broth,expression of the peptibody was induced by addition ofN-(3-oxo-hexanoyl)-homoserine lactone at 50 ng/ml and after 4-6 hours ofshaking at 37 C the cells were centrifuged and the cell paste stored at−20 C.

The following gives for each example the cloning vector and the set ofoligonucleotides used for constructing each fusion protein. Also shownis a DNA/protein map.

Bacterial expression of Fc-L-ShK[1-35] inhibitor of Kv1.3. The methodsto clone and express the peptibody in bacteria are described above. Thevector used was pAMG21ampR-Fc-Pep and the oligos listed below were usedto generate a duplex (see below) for cloning and expression in bacteriaof Fc-L-ShK[1-35].

Oligos used to form the duplex:

//SEQ ID NO: 669 TGGTTCCGGTGGTGGTGGTTCCCGTTCCTGCATCGACACCAT;//SEQ ID NO: 670 CCCGAAATCCCGTTGCACCGCTTTCCAGTGCAAACACTCCATGAAATACCGTCTGTCCTTCTGCCGTAAAACCTGCGGTACCTGC; //SEQ ID NO: 671CTTAGCAGGTACCGCAGGTTTTACGGCAGAAGGACAGACGGT; //SEQ ID NO: 672ATTTCATGGAGTGTTTGCACTGGAAAGCGGTGCAACGGGATTTCGGGATGGTGTCGATGCAGGAACGGGAACCACCACCACCGGA;

The oligo duplex is shown below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen. Purification of bacterially expressedFc-L10-ShK(1-35) is further described in Example 38 herein below.

Example 4 Fc-L-ShK[2-35] Bacterial Expression

Bacterial expression of Fc-L-ShK[2-35]. The methods to clone and expressthe peptibody in bacteria are described in Example 3. The vector usedwas pAMG21ampR-Fc-Pep and the oligos listed below were used to generatea duplex (see below) for cloning and expression in bacteria ofFc-L-ShK[2-35].

Oligos used to form duplex are shown below:

//SEQ ID NO: 676 TGGTTCCGGTGGTGGTGGTTCCTGCATCGACACCATCCCGAAATCCCGTTGCACCGCTTTCCAGTGCAAACACTCCATGAAAT; //SEQ ID NO: 677ACCGTCTGTCCTTCTGCCGTAAAACCTGCGGTACCTGC; //SEQ ID NO: 678CTTAGCAGGTACCGCAGGTTTTACGGCAGAAGGACAGACGGTATTTCATGGAGTGTTTGCACTGGAAAGCGGTGCAACGGGA; //SEQ ID NO: 679TTTCGGGATGGTGTCGATGCAGGAACCACCACCACCGGA;

The oligo duplex formed is shown below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen. Purification of bacterially expressedFc-L10-ShK(2-35) is further described in Example 39 herein below.

Example 5 Fc-L-HmK Bacterial Expression

Bacterial expression of Fc-L-HmK. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-HmK.

Oligos used to form duplex are shown below:

//SEQ ID NO: 690 TGGTTCCGGTGGTGGTGGTTCCCGTACCTGCAAAGACCTGAT;SEQ ID NO: 692 CCCGGTTTCCGAATGCACCGACATCCGTTGCCGTACCTCCATGAAATACCGTCTGAACCTGTGCCGTAAAACCTGCGGTTCCTGC; //SEQ ID NO: 693CTTAGCAGGAACCGCAGGTTTTACGGCACAGGTTCAGACGGT; //SEQ ID NO: 694ATTTCATGGAGGTACGGCAACGGATGTCGGTGCATTCGGAAACCGGGATCAGGTCTTTGCAGGTACGGGAACCACCACCACCGGA;

The oligo duplex formed is shown below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 6 Fc-L-KTX1 Bacterial Expression

Bacterial expression of Fc-L-KTX1. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-KTX1.

Oligos used to form duplex are shown below:

//SEQ ID NO: 698 TGGTTCCGGTGGTGGTGGTTCCGGTGTTGAAATCAACGTTAAATGCT;//SEQ ID NO: 699 CCGGTTCCCCGCAGTGCCTGAAACCGTGCAAAGACGCTGGTATGCGTTTCGGTAAATGCATGAACCGTAAATGCCACTGCACCCCGAAA; //SEQ ID NO: 700CTTATTTCGGGGTGCAGTGGCATTTACGGTTCATGCATTTACCGAAA; //SEQ ID NO: 701CGCATACCAGCGTCTTTGCACGGTTTCAGGCACTGCGGGGAACCGGAGCATTTAACGTTGATTTCAACACCGGAACCACCACCACCGGA;

The oligo duplex formed is shown below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Purification and refolding of Fc-L-KTX1 expressed in bacteria. Frozen,E. coli paste (28 g) was combined with 210 ml of room temperature 50 mMtris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen eggwhite lysozyme. The suspended paste was passed through a chilledmicrofluidizer twice at 12,000 PSI. The cell lysate was then centrifugedat 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200ml 1% deoxycholic acid using a tissue grinder and then centrifuged at22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 mlwater using a tissue grinder and then centrifuged at 22,000 g for 20 minat 4° C. The pellet (4.8 g) was then dissolved in 48 ml 8 M guanidineHCl, 50 mM tris HCl, pH 8.0. The dissolved pellet was then reduced byadding 30 μl 1 M dithiothreitol to 3 ml of the solution and incubatingat 37° C. for 30 minutes. The reduced pellet solution was thencentrifuged at 14,000 g for 5 min at room temperature, and then 2.5 mlof the supernatant was transferred to 250 ml of the refolding buffer (2M urea, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1 mM cystamine HCl,4 mM cysteine, pH 8.5) at 4° C. with vigorous stirring. The stirringrate was then slowed and the incubation was continued for 2 days at 4°C. The refolding solution was then filtered through a 0.22 μm celluloseacetate filter and stored at 4° C. for 3 days.

The stored refold was then diluted with 1 L of water and the pH wasadjusted to 7.5 using 1 M H₃PO₄. The pH adjusted material was thenloaded on to a 10 ml Amersham SP-HP HiTrap column at 10 ml/min inS-Buffer A (20 mM NaH₂PO₄, pH 7.3) at 7° C. The column was then washedwith several column volumes of S-Buffer A, followed by elution with alinear gradient from 0% to 60% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH7.3) followed by a step to 100% S-Buffer B at 5 ml/min 7° C. Fractionswere then analyzed using a Coomassie brilliant blue stained tris-glycine4-20% SDS-PAGE, and the fractions containing the desired product werepooled based on these data (45 ml). The pool was then loaded on to a 1ml Amersham rProtein A HiTrap column in PBS at 2 ml/min 7° C. Thencolumn was then washed with several column volumes of PBS and elutedwith 100 mM glycine pH 3.0. To the elution peak (2.5 ml), 62.5 μl 2 Mtris base was added, and then the pH adjusted material was filteredthough a Pall Life Sciences Acrodisc with a 0.22 μm, 25 mm Mustang Emembrane at 2 ml/min room temperature.

A spectral scan was then conducted on 20 μl of the combined pool dilutedin 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 28C).The concentration of the filtered material was determined to be 2.49mg/ml using a calculated molecular mass of 30,504 g/mol and extinctioncoefficient of 35,410 M⁻¹ cm⁻¹. The purity of the filtered material wasthen assessed using a Coomassie brilliant blue stained tris-glycine4-20% SDS-PAGE (FIG. 28A). The endotoxin level was then determined usinga Charles River Laboratories Endosafe-PTS system (0.05-5 EU/mlsensitivity) using a 50-fold dilution of the sample in Charles RiversEndotoxin Specific Buffer BG120 yielding a result of <1 EU/mg protein.The macromolecular state of the product was then determined using sizeexclusion chromatography on 45 μg of the product injected on to aPhenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mMNaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 28B).The product was then subject to mass spectral analysis by diluting 1 μlof the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05%trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl)was spotted onto a MALDI sample plate. The sample was allowed to drybefore being analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses (FIG. 28D) and these studiesconfirmed the integrity of the purified peptibody, within experimentalerror. The product was then stored at −80° C.

Purified Fc-L-KTX1 blocked the human Kv1.3 current in a dose-dependentfashion (FIG. 32A and FIG. 32B) by electrophysiology (method was asdescribed in Example 36).

Example 7 Fc-L-HsTx1 Bacterial Expression

Bacterial expression of Fc-L-HsT1. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-HsTx1.

Oligos used to form duplex are shown below:

//SEQ ID NO: 705 TGGTTCCGGTGGTGGTGGTTCCGCTTCCTGCCGTACCCCGAAAGAC;//SEQ ID NO: 706 TGCGCTGACCCGTGCCGTAAAGAAACCGGTTGCCCGTACGGTAAATGCATGAACCGTAAATGCAAATGCAACCGTTGC; //SEQ ID NO: 707CTTAGCAACGGTTGCATTTGCATTTACGGTTCATGCATTTACCGTACG; //SEQ ID NO: 708GGCAACCGGTTTCTTTACGGCACGGGTCAGCGCAGTCTTTCGGGGTACGGCAGGAAGCGGAACCACCACCACCGGA;

The duplex formed by the oligos above is shown below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 8 Fc-L-MgTx Bacterial Expression

Bacterial expression of Fc-L-MgTx. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-MgTx.

Oligos used to form duplex are shown below:

//SEQ ID NO: 712 TGGTTCCGGTGGTGGTGGTTCCACCATCATCAACGTTAAATGCACCTC;//SEQ ID NO: 713 CCCGAAACAGTGCCTGCCGCCGTGCAAAGCTCAGTTCGGTCAGTCCGCTGGTGCTAAATGCATGAACGGTAAATGCAAATGCTACCCGCAC; //SEQ ID NO: 714CTTAGTGCGGGTAGCATTTGCATTTACCGTTCATGCATTTAGCACCAG; //SEQ ID NO: 715CGGACTGACCGAACTGAGCTTTGCACGGCGGCAGGCACTGTTTCGGGGAGGTGCATTTAACGTTGATGATGGTGGAACCACCACCACCGGA;

The oligos above were used to form the duplex shown below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 9 Fc-L-AgTx2 Bacterial Expression

Bacterial expression of Fc-L-AgTx2. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-AgTx2.

Oligos used to form duplex are shown below:

//SEQ ID NO: 719 TGGTTCCGGTGGTGGTGGTTCCGGTGTTCCGATCAACGTTTCCTGCACCG GT;//SEQ ID NO: 720 TCCCCGCAGTGCATCAAACCGTGCAAAGACGCTGGTATGCGTTTCGGTAAATGCATGAACCGTAAATGCCACTGCACCCCGAAA; //SEQ ID NO: 721CTTATTTCGGGGTGCAGTGGCATTTACGGTTCATGCATTTACCGAAACGC ATA; //SEQ ID NO: 722CCAGCGTCTTTGCACGGTTTGATGCACTGCGGGGAACCGGTGCAGGAAACGTTGATCGGAACACCGGAACCACCACCACCGGA;

The oligos listed above were used to form the duplex shown below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Refolding and purification of Fc-L-AgTx2 expressed in bacteria. Frozen,E. coli paste (15 g) was combined with 120 ml of room temperature 50 mMtris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen eggwhite lysozyme. The suspended paste was passed through a chilledmicrofluidizer twice at 12,000 PSI. The cell lysate was then centrifugedat 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200ml 1% deoxycholic acid using a tissue grinder and then centrifuged at22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 mlwater using a tissue grinder and then centrifuged at 22,000 g for 20 minat 4° C. The pellet (4.6 g) was then dissolved in 46 ml 8 M guanidineHCl, 50 mM tris HCl, pH 8.0. The dissolved pellet was then reduced byadding 30 μl 1 M dithiothreitol to 3 ml of the solution and incubatingat 37° C. for 30 minutes. The reduced pellet solution was thencentrifuged at 14,000 g for 5 min at room temperature, and then 2.5 mlof the supernatant was transferred to 250 ml of the refolding buffer (2M urea, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1 mM cystamine HCl,4 mM cysteine, pH 9.5) at 4° C. with vigorous stirring. The stirringrate was then slowed and the incubation was continued for 2 days at 4°C. The refolding solution was then filtered through a 0.22 μm celluloseacetate filter and stored at −70° C.

The stored refold was defrosted and then diluted with 1 L of water andthe pH was adjusted to 7.5 using 1 M H₃PO₄. The pH adjusted material wasthen filtered through a 0.22 μm cellulose acetate filter and loaded onto a 10 ml Amersham SP-HP HiTrap column at 10 ml/min in S-Buffer A (20mM NaH₂PO₄, pH 7.3) at 7° C. The column was then washed with severalcolumn volumes of S-Buffer A, followed by elution with a linear gradientfrom 0% to 60% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.3) followed bya step to 100% S-Buffer B at 5 ml/min 7° C. Fractions were then analyzedusing a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE,and the fractions containing the desired product were pooled based onthese data (15 ml). The pool was then loaded on to a 1 ml AmershamrProtein A HiTrap column in PBS at 2 ml/min 7° C. Then column was thenwashed with several column volumes of 20 mM NaH₂PO₄ pH 6.5, 1 M NaCl andeluted with 100 mM glycine pH 3.0. To the elution peak (1.5 ml), 70 μl 1M tris HCl pH 8.5 was added, and then the pH-adjusted material wasfiltered though a 0.22 μm cellulose acetate filter.

A spectral scan was then conducted on 20 μl of the combined pool dilutedin 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 29C).The concentration of the filtered material was determined to be 1.65mg/ml using a calculated molecular mass of 30,446 g/mol and extinctioncoefficient of 35,410 M⁻¹ cm⁻¹. The purity of the filtered material wasthen assessed using a Coomassie brilliant blue stained tris-glycine4-20% SDS-PAGE (FIG. 29A). The endotoxin level was then determined usinga Charles River Laboratories Endosafe-PTS system (0.05-5 EU/mlsensitivity) using a 33-fold dilution of the sample in Charles RiversEndotoxin Specific Buffer BG120 yielding a result of <4 EU/mg protein.The macromolecular state of the product was then determined using sizeexclusion chromatography on 20 μg of the product injected on to aPhenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mMNaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 29D).The product was then subject to mass spectral analysis by diluting 1 μlof the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05%trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl)was spotted onto a MALDI sample plate. The sample was allowed to drybefore being analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses (FIG. 29E) and these studiesconfirmed the integrity of the purified peptibody, within experimentalerror. The product was then stored at −80° C.

Example 10 Fc-L-OSK1 Bacterial Expression

Bacterial expression of Fc-L-OSK1. The methods used to clone and expressthe peptibody in bacteria were as described in Example 3. The vectorused was pAMG21ampR-Fc-Pep and the oligos listed below were used togenerate a duplex (see below) for cloning and expression in bacteria ofFc-L-OSK1.

Oligos used to form duplex are shown below:

//SEQ ID NO: 726 TGGTTCCGGTGGTGGTGGTTCCGGTGTTATCATCAACGTTAAATGCAAAATCTCCCGTCAGTGCCTGGAACCGTGCAAAAAAG; //SEQ ID NO: 727CTGGTATGCGTTTCGGTAAATGCATGAACGGTAAATGCCACTGCACCCCG AAA; //SEQ ID NO: 728CTTATTTCGGGGTGCAGTGGCATTTACCGTTCATGCATTTACCGAAACGCATACCAGCTTTTTTGCACGGTTCCAGGCACTGA; //SEQ ID NO: 729CGGGAGATTTTGCATTTAACGTTGATGATAACACCGGAACCACCACCACC GGA;

The oligos shown above were used to form the duplex below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen for later use. Purification of Fc-L10-OSK1 fromE. coli paste is described in Example 40 herein below.

Example 11 Fc-L-OSK1(E16K, K20D) Bacterial Expression

Bacterial expression of Fc-L-OSK1(E16K, K20D). The methods to clone andexpress the peptibody in bacteria are described in Example 3. The vectorused was pAMG21ampR-Fc-Pep the oligos listed below were used to generatea duplex (see below) for cloning and expression in bacteria ofFc-L-OSK1(E16K,K20D).

Oligos used to form duplex are shown below:

//SEQ ID NO: 733 TGGTTCCGGTGGTGGTGGTTCCGGTGTTATCATCAACGTTAAATGCAAAATCTCCCGTCAGTGCCTGAAACCGTGCAAAGACG; //SEQ ID NO: 734CTGGTATGCGTTTCGGTAAATGCATGAACGGTAAATGCCACTGCACCCCG AAA; //SEQ ID NO: 735CTTATTTCGGGGTGCAGTGGCATTTACCGTTCATGCATTTACCGAAACGCATACCAGCGTCTTTGCACGGTTTCAGGCACTGA; //SEQ ID NO: 736CGGGAGATTTTGCATTTAACGTTGATGATAACACCGGAACCACCACCACC GGA;

The oligos shown above were used to form the duplex below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen for later use.

Example 12 Fc-L-Anuroctoxin Bacterial Expression

Bacterial expression of Fc-L-Anuroctoxin. The methods to clone andexpress the peptibody in bacteria are described in Example 3. The vectorused was pAMG21ampR-Fc-Pep and the oligos listed below were used togenerate a duplex (see below) for cloning and expression in bacteria ofFc-L-Anuroctoxin.

Oligos used to form duplex are shown below:

//SEQ ID NO: 740 TGGTTCCGGTGGTGGTGGTTCCAAAGAATGCACCGGTCCGCAGCACTGCACCAACTTCTGCCGTAAAAACAAATGCACCCACG; //SEQ ID NO: 741GTAAATGCATGAACCGTAAATGCAAATGCTTCAACTGCAAA; //SEQ ID NO: 742CTTATTTGCAGTTGAAGCATTTGCATTTACGGTTCATGCATTTACCGTGGGTGCATTTGTTTTTACGGCAGAAGTTGGTGCAG; //SEQ ID NO: 743TGCTGCGGACCGGTGCATTCTTTGGAACCACCACCACCGGA;

The oligos shown above were used to form the duplex below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 13 Fc-L-Noxiustoxin Bacterial Expression

Bacterial expression of Fc-L-Noxiustoxin or Fc-L-NTX. The methods toclone and express the peptibody in bacteria are described in Example 3.The vector used was pAMG21ampR-Fc-Pep and the oligos listed below wereused to generate a duplex (see below) for cloning and expression inbacteria of Fc-L-NTX.

Oligos used to form duplex are shown below:

//SEQ ID NO: 747 TGGTTCCGGTGGTGGTGGTTCCACCATCATCAACGTTAAATGCACCTCCCCGAAACAGTGCTCCAAACCGTGCAAAGAACTGT; //SEQ ID NO: 748ACGGTTCCTCCGCTGGTGCTAAATGCATGAACGGTAAATGCAAATGCTAC AACAAC;//SEQ ID NO: 749 CTTAGTTGTTGTAGCATTTGCATTTACCGTTCATGCATTTAGCACCAGCGGAGGAACCGTACAGTTCTTTGCACGGTTTGGAG; //SEQ ID NO: 750CACTGTTTCGGGGAGGTGCATTTAACGTTGATGATGGTGGAACCACCACC ACCGGA;

The oligos shown above were used to form the duplex below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 14 Fc-L-Pi2 Bacterial Expression

Bacterial expression of Fc-L-Pi2. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-Pi2.

Oligos used to form duplex are shown below:

//SEQ ID NO: 754 TGGTTCCGGTGGTGGTGGTTCCACCATCTCCTGCACCAACCCG;//SEQ ID NO: 755 AAACAGTGCTACCCGCACTGCAAAAAAGAAACCGGTTACCCGAACGCTAAATGCATGAACCGTAAATGCAAATGCTTCGGTCGT; //SEQ ID NO: 756CTTAACGACCGAAGCATTTGCATTTACGGTTCATGCATTTAGCG; //SEQ ID NO: 757TTCGGGTAACCGGTTTCTTTTTTGCAGTGCGGGTAGCACTGTTTCGGGTTGGTGCAGGAGATGGTGGAACCACCACCACCGGA;

The oligos above were used to form the duplex below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 15 ShK[1-35]-L-Fc Bacterial Expression

Bacterial expression of ShK[1-35]-L-Fc. The methods to clone and expressthe peptibody in bacteria are described in Example 3. The vector usedwas pAMG21ampR-Pep-Fc and the oligos listed below were used to generatea duplex (see below) for cloning and expression in bacteria ofShK[1-35]-L-Fc.

Oligos used to form duplex are shown below:

//SEQ ID NO: 761 TATGCGTTCTTGTATTGATACTATTCCAAAATCTCGTTGTACTGCTTTTCAATGTAAACATTCTATGAAATATCGTCTTTCTT; //SEQ ID NO: 762TTTGTCGTAAAACTTGTGGTACTTGTTCTGGTGGTGGTGGTTCT; //SEQ ID NO: 763CACCAGAACCACCACCACCAGAACAAGTACCACAAGTTTTACGACAAAAAGAAAGACGATATTTCATAGAATGTTTACATTGA; //SEQ ID NO: 764AAAGCAGTACAACGAGATTTTGGAATAGTATCAATACAAGAACG;

The oligos shown above were used to form the duplex shown below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen. Purification of met-ShK[1-35]-Fc was asdescribed in Example 51 herein below.

Example 16 ShK[2-35]-L-Fc Bacterial Expression

Bacterial expression of ShK[2-35]-L-Fc. The methods to clone and expressthe peptibody in bacteria are described in Example 3. The vector usedwas pAMG21ampR-Pep-Fc and the oligos listed below were used to generatea duplex (see below) for cloning and expression in bacteria ofShK[2-35]-L-Fc.

Oligos used to form duplex are shown below:

//SEQ ID NO: 768 TATGTCTTGTATTGATACTATTCCAAAATCTCGTTGTACTGCTTTTCAATGTAAACATTCTATGAAATATCGTCTTTCTT; //SEQ ID NO: 769TTTGTCGTAAAACTTGTGGTACTTGTTCTGGTGGTGGTGGTTCT; //SEQ ID NO: 770CACCAGAACCACCACCACCAGAACAAGTACCACAAGTTTTACGACAAAAAGAAAGACGATATTTCATAGAATGTTTACATTGA; SEQ ID NO: 771AAAGCAGTACAACGAGATTTTGGAATAGTATCAATACAAGA;

The oligos above were used to form the duplex shown below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen. Purification of the ShK[2-35]-Fc was asdescribed in Example 50 herein below.

Example 17 Fc-L-ChTx Bacterial Expression

Bacterial expression of Fc-L-ChTx. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-ChTx.

Oligos used to form duplex are shown below:

//SEQ ID NO: 775 TGGTTCCGGTGGTGGTGGTTCCCAGTTCACCAACGTT; //SEQ ID NO: 776TCCTGCACCACCTCCAAAGAATGCTGGTCCGTTTGCCAGCGTCTGCACAACACCTCCCGTGGTAAATGCATGAACAAAAAATGCCGTTGCTACTCC; //SEQ ID NO: 777CTTAGGAGTAGCAACGGCATTTTTTGTTCATGCATTTA; //SEQ ID NO: 778CCACGGGAGGTGTTGTGCAGACGCTGGCAAACGGACCAGCATTCTTTGGAGGTGGTGCAGGAAACGTTGGTGAACTGGGAACCACCACCACCGGA;

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 18 Fc-L-MTX Bacterial Expression

Bacterial expression of Fc-L-MTX. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-MTX.

Oligos used to form duplex are shown below:

//SEQ ID NO: 782 TGGTTCCGGTGGTGGTGGTTCCGTTTCCTGCACCGGT; //SEQ ID NO: 783TCCAAAGACTGCTACGCTCCGTGCCGTAAACAGACCGGTTGCCCGAACGCTAAATGCATCAACAAATCCTGCAAATGCTACGGTTGC; //SEQ ID NO: 784CTTAGCAACCGTAGCATTTGCAGGATTTGTTGATGCAT; //SEQ ID NO: 785TTAGCGTTCGGGCAACCGGTCTGTTTACGGCACGGAGCGTAGCAGTCTTTGGAACCGGTGCAGGAAACGGAACCACCACCACCGGA;

The oligos above were used to form the duplex shown below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 19 Fc-L-ChTx (K32E) Bacterial Expression

Bacterial expression of Fc-L-ChTx (K32E). The methods to clone andexpress the peptibody in bacteria are described in Example 3. The vectorused was pAMG21ampR-Fc-Pep and the oligos listed below were used togenerate a duplex (see below) for cloning and expression in bacteria ofFc-L-ChTx (K32E).

Oligos used to form duplex are shown below:

//SEQ ID NO: 789 TGGTTCCGGTGGTGGTGGTTCCCAGTTCACCAACGTTTCCTG;//SEQ ID NO: 790 CACCACCTCCAAAGAATGCTGGTCCGTTTGCCAGCGTCTGCACAACACCTCCCGTGGTAAATGCATGAACAAAGAATGCCGTTGCTACTCC; //SEQ ID NO: 791CTTAGGAGTAGCAACGGCATTCTTTGTTCATGCATTTACCACG; //SEQ ID NO: 792GGAGGTGTTGTGCAGACGCTGGCAAACGGACCAGCATTCTTTGGAGGTGGTGCAGGAAACGTTGGTGAACTGGGAACCACCACCACCGGA;

The oligos shown above were used to form the duplex below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 20 Fc-L-Apamin Bacterial Expression

Bacterial expression of Fc-L-Apamin. The methods to clone and expressthe peptibody in bacteria are described in Example 3. The vector usedwas pAMG21ampR-Fc-Pep and the oligos listed below were used to generatea duplex (see below) for cloning and expression in bacteria ofFc-L-Apamin.

Oligos used to form duplex are shown below:

//SEQ ID NO: 796 TGGTTCCGGTGGTGGTGGTTCCTGCAACTGCAAAGCTCCGGAAACCGCTCTGTGCGCTCGTCGTTGCCAGCAGCACGGT; //SEQ ID NO: 797CTTAACCGTGCTGCTGGCAACGACGAGCGCACAGAGCGGTTTCCGGAGCTTTGCAGTTGCAGGAACCACCACCACCGGA;

The oligos above were used to form the duplex shown below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 21 Fc-L-Scyllatoxin Bacterial Expression

Bacterial expression of Fc-L-Scyllatoxin or Fc-L-ScyTx. The methods toclone and express the peptibody in bacteria are described in Example 3.The vector used was pAMG21ampR-Fc-Pep and the oligos listed below wereused to generate a duplex (see below) for cloning and expression inbacteria of Fc-L-ScyTx.

Oligos used to form duplex are shown below:

//SEQ ID NO: 801 TGGTTCCGGTGGTGGTGGTTCCGCTTTCTGCAACCTGCG;//SEQ ID NO: 802 TATGTGCCAGCTGTCCTGCCGTTCCCTGGGTCTGCTGGGTAAATGCATCGGTGACAAATGCGAATGCGTTAAACAC; //SEQ ID NO: 803CTTAGTGTTTAACGCATTCGCATTTGTCACCGATGCATTT; //SEQ ID NO: 804ACCCAGCAGACCCAGGGAACGGCAGGACAGCTGGCACATACGCAGGTTGCAGAAAGCGGAACCACCACCACCGGA;

The oligos above were used to form the duplex below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 22 Fc-L-IbTx Bacterial Expression

Bacterial expression of Fc-L-lbTx. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-lbTx.

Oligos used to form duplex are shown below:

//SEQ ID NO: 808 TGGTTCCGGTGGTGGTGGTTCCCAGTTCACCGACGTTGACTGCTCCGT;//SEQ ID NO: 809 TTCCAAAGAATGCTGGTCCGTTTGCAAAGACCTGTTCGGTGTTGACCGTGGTAAATGCATGGGTAAAAAATGCCGTTGCTACCAG; //SEQ ID NO: 810CTTACTGGTAGCAACGGCATTTTTTACCCATGCATTTACCACGGTCAA; //SEQ ID NO: 811CACCGAACAGGTCTTTGCAAACGGACCAGCATTCTTTGGAAACGGAGCAGTCAACGTCGGTGAACTGGGAACCACCACCACCGGA;

The oligos above were used to form the duplex below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 23 Fc-L-HaTx1 Bacterial Expression

Bacterial expression of Fc-L-HaTx1. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-HaTx1.

Oligos used to form duplex are shown below:

//SEQ ID NO: 815 TGGTTCCGGTGGTGGTGGTTCCGAATGCCGTTACCTGTTCGGTGGTTG;//SEQ ID NO: 816 CAAAACCACCTCCGACTGCTGCAAACACCTGGGTTGCAAATTCCGTGACAAATACTGCGCTTGGGACTTCACCTTCTCC; //SEQ ID NO: 817CTTAGGAGAAGGTGAAGTCCCAAGCGCAGTATTTGTCACGGAATTTGC; //SEQ ID NO: 818AACCCAGGTGTTTGCAGCAGTCGGAGGTGGTTTTGCAACCACCGAACAGGTAACGGCATTCGGAACCACCACCACCGGA;

The oligos above were used to form the duplex below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Refolding and purification of Fc-L-HaTx1 expressed in bacteria. Frozen,E. coli paste (13 g) was combined with 100 ml of room temperature 50 mMtris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen eggwhite lysozyme. The suspended paste was passed through a chilledmicrofluidizer twice at 12,000 PSI. The cell lysate was then centrifugedat 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200ml 1% deoxycholic acid using a tissue grinder and then centrifuged at22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 mlwater using a tissue grinder and then centrifuged at 22,000 g for 20 minat 4° C. The pellet (2.6 g) was then dissolved in 26 ml 8 M guanidineHCl, 50 mM tris HCl, pH 8.0. The dissolved pellet was then reduced byadding 30 μl 1 M dithiothreitol to 3 ml of the solution and incubatingat 37° C. for 30 minutes. The reduced pellet solution was thencentrifuged at 14,000 g for 5 min at room temperature, and then 2.5 mlof the supernatant was transferred to 250 ml of the refolding buffer (2M urea, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1 mM cystamine HCl,4 mM cysteine, pH 8.5) at 4° C. with vigorous stirring. The stirringrate was then slowed and the incubation was continued for 2 days at 4°C. The refolding solution was then filtered through a 0.22 μm celluloseacetate filter and stored at −70° C.

The stored refold was defrosted and then diluted with 1 L of water andthe pH was adjusted to 7.5 using 1 M H₃PO₄. The pH adjusted material wasthen filtered through a 0.22 μm cellulose acetate filter and loaded onto a 10 ml Amersham SP-HP HiTrap column at 10 ml/min in S-Buffer A (20mM NaH₂PO₄, pH 7.3) at 7° C. The column was then washed with severalcolumn volumes of S-Buffer A, followed by elution with a linear gradientfrom 0% to 60% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.3) followed bya step to 100% S-Buffer B at 5 ml/min 7° C. Fractions were then analyzedusing a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE,and the fractions containing the desired product were pooled based onthese data (15 ml). The pool was then loaded on to a 1 ml AmershamrProtein A HiTrap column in PBS at 2 ml/min 7° C. Then column was thenwashed with several column volumes of 20 mM NaH₂PO₄ pH 6.5, 1 M NaCl andeluted with 100 mM glycine pH 3.0. To the elution peak (1.4 ml), 70 μl 1M tris HCl pH 8.5 was added, and then the pH adjusted material wasfiltered though a 0.22 μm cellulose acetate filter.

A spectral scan was then conducted on 20 μl of the combined pool dilutedin 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 29F).The concentration of the filtered material was determined to be 1.44mg/ml using a calculated molecular mass of 30,469 g/mol and extinctioncoefficient of 43,890 M⁻¹ cm⁻¹. The purity of the filtered material wasthen assessed using a Coomassie brilliant blue stained tris-glycine4-20% SDS-PAGE (FIG. 29B). The endotoxin level was then determined usinga Charles River Laboratories Endosafe-PTS system (0.05-5 EU/mlsensitivity) using a 33-fold dilution of the sample in Charles RiversEndotoxin Specific Buffer BG120 yielding a result of <4 EU/mg protein.The macromolecular state of the product was then determined using sizeexclusion chromatography on 20 μg of the product injected on to aPhenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mMNaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 29G).The product was then subject to mass spectral analysis by diluting 1 μlof the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05%trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl)was spotted onto a MALDI sample plate. The sample was allowed to drybefore being analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from ˜200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses (FIG. 29H) and these studiesconfirmed the integrity of the purified peptibody, within experimentalerror. The product was then stored at −80° C.

Example 24 Fc-L-PaTx2 Bacterial Expression

Bacterial expression of Fc-L-PaTx2. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-PaTx2.

Oligos used to form duplex are shown below:

//SEQ ID NO: 822 TGGTTCCGGTGGTGGTGGTTCCTACTGCCAGAAATGGA;//SEQ ID NO: 823 TGTGGACCTGCGACGAAGAACGTAAATGCTGCGAAGGTCTGGTTTGCCGTCTGTGGTGCAAACGTATCATCAACATG; //SEQ ID NO: 824CTTACATGTTGATGATACGTTTGCACCACAGACGGCAAA; //SEQ ID NO: 825CCAGACCTTCGCAGCATTTACGTTCTTCGTCGCAGGTCCACATCCATTTCTGGCAGTAGGAACCACCACCACCGGA;

The oligos above were used to form the duplex below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 25 Fc-L-wGVIA Bacterial Expression

Bacterial expression of Fc-L-wGVIA. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-wGVIA.

Oligos used to form duplex are shown below:

//SEQ ID NO: 829 TGGTTCCGGTGGTGGTGGTTCCTGCAAATCCCCGGGTT; SEQ ID NO: 830CCTCCTGCTCCCCGACCTCCTACAACTGCTGCCGTTCCTGCAACCCGTAC ACCAAACGTTGCTACGGT;//SEQ ID NO: 831 CTTAACCGTAGCAACGTTTGGTGTACGGGTTGCAGGAA;//SEQ ID NO: 832 CGGCAGCAGTTGTAGGAGGTCGGGGAGCAGGAGGAACCCGGGGATTTGCAGGAACCACCACCACCGGA;

The oligos above were used to form the duplex below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 26 Fc-L-ωMVIIA Bacterial Expression

Bacterial expression of Fc-L-ωMVIIA. The methods to clone and expressthe peptibody in bacteria are described in Example 3. The vector usedwas pAMG21ampR-Fc-Pep and the oligos listed below were used to generatea duplex (see below) for cloning and expression in bacteria ofFc-L-ωMVIIA.

Oligos used to form duplex are shown below:

//SEQ ID NO: 836 TGGTTCCGGTGGTGGTGGTTCCTGCAAAGGTAAA; //SEQ ID NO: 837GGTGCTAAATGCTCCCGTCTGATGTACGACTGCTGCACCGGTTCCTGCCG TTCCGGTAAATGCGGT;//SEQ ID NO: 838 CTTAACCGCATTTACCGGAACGGCAGGAACCGGT; //SEQ ID NO: 839GCAGCAGTCGTACATCAGACGGGAGCATTTAGCACCTTTACCTTTGCAGG AACCACCACCACCGGA;

The oligos above were used to form the duplex below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 27 Fc-L-PtuI Bacterial Expression

Bacterial expression of Fc-L-Ptu1. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-Ptu1.

Oligos used to form duplex are shown below:

//SEQ ID NO: 843 TGGTTCCGGTGGTGGTGGTTCCGCTGAAAAAGACTGCATC;//SEQ ID NO: 844 GCTCCGGGTGCTCCGTGCTTCGGTACCGACAAACCGTGCTGCAACCCGCGTGCTTGGTGCTCCTCCTACGCTAACAAATGCCTG; //SEQ ID NO: 845CTTACAGGCATTTGTTAGCGTAGGAGGAGCACCAAGCACG; //SEQ ID NO: 846CGGGTTGCAGCACGGTTTGTCGGTACCGAAGCACGGAGCACCCGGAGCGATGCAGTCTTTTTCAGCGGAACCACCACCACCGGA;

The oligos above were used to form the duplex below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 28 Fc-L-ProTx1 Bacterial Expression

Bacterial expression of Fc-L-ProTx1. The methods to clone and expressthe peptibody in bacteria are described in Example 3. The vector usedwas pAMG21ampR-Fc-Pep and the oligos listed below were used to generatea duplex (see below) for cloning and expression in bacteria ofFc-L-ProTx1.

Oligos used to form duplex are shown below:

//SEQ ID NO: 850 TGGTTCCGGTGGTGGTGGTTCCGAATGCCGTTACTGGCTGG;//SEQ ID NO: 851 GTGGTTGCTCCGCTGGTCAGACCTGCTGCAAACACCTGGTTTGCTCCCGTCGTCACGGTTGGTGCGTTTGGGACGGTACCTTCTCC; //SEQ ID NO: 852CTTAGGAGAAGGTACCGTCCCAAACGCACCAACCGTGACGA; //SEQ ID NO: 853CGGGAGCAAACCAGGTGTTTGCAGCAGGTCTGACCAGCGGAGCAACCACCCAGCCAGTAACGGCATTCGGAACCACCACCACCGGA;

The oligos above were used to form the duplex below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 29 Fc-L-BeKM1 Bacterial Expression

Bacterial expression of Fc-L-BeKM1. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-BeKM1.

Oligos used to form duplex are shown below:

//SEQ ID NO: 857 TGGTTCCGGTGGTGGTGGTTCCCGTCCGACCGACATCAAATG;//SEQ ID NO: 858 CTCCGAATCCTACCAGTGCTTCCCGGTTTGCAAATCCCGTTTCGGTAAAACCAACGGTCGTTGCGTTAACGGTTTCTGCGACTGCTTC; //SEQ ID NO: 859CTTAGAAGCAGTCGCAGAAACCGTTAACGCAACGACCGTTGG; //SEQ ID NO: 860TTTTACCGAAACGGGATTTGCAAACCGGGAAGCACTGGTAGGATTCGGAGCATTTGATGTCGGTCGGACGGGAACCACCACCACCGGA;

The oligos above were used to form the duplex below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 30 Fc-L-CTX Bacterial Expression

Bacterial expression of Fc-L-CTX. The methods to clone and express thepeptibody in bacteria are described in Example 3. The vector used waspAMG21ampR-Fc-Pep and the oligos listed below were used to generate aduplex (see below) for cloning and expression in bacteria of Fc-L-CTX.

Oligos used to form duplex are shown below:

//SEQ ID NO: 864 TGGTTCCGGTGGTGGTGGTTCCATGTGCATGCCGTGCTTCAC;//SEQ ID NO: 865 CACCGACCACCAGATGGCTCGTAAATGCGACGACTGCTGCGGTGGTAAAGGTCGTGGTAAATGCTACGGTCCGCAGTGCCTGTGCCGT; //SEQ ID NO: 866CTTAACGGCACAGGCACTGCGGACCGTAGCATTTACCACGAC; //SEQ ID NO: 867CTTTACCACCGCAGCAGTCGTCGCATTTACGAGCCATCTGGTGGTCGGTGGTGAAGCACGGCATGCACATGGAACCACCACCACCGGA;

The oligos above were used to form the duplex below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 31 N-Terminally PEGylated-Des-Arg1-ShK

Peptide Synthesis of reduced Des-Arq1-ShK. Des-Arg1-ShK, having thesequence

(Peptide 1, SEQ ID NO: 92) SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTCwas synthesized in a stepwise manner on a Symphony™ multi-peptidesynthesizer by solid-phase peptide synthesis (SPPS) using2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU)/N-methyl morpholine (NMM)/N,N-dimethyl-formamide (DMF) couplingchemistry at 0.1 mmol equivalent resin scale on Tentagel™-S PHBFmoc-Cys(Trt)-resin. N-alpha-(9-fluorenylmethyloxycarbonyl)- andside-chain protected amino acids were purchased from Midwest BiotechIncorporated. Fmoc-Cys(Trt)-Tentagel™ resin was purchased from Fluka.The following side-chain protection strategy was employed: Asp(O^(t)Bu),Arg(Pbf), Cys(Trt), Gln(Trt), His(Trt), Lys(N^(ε)-Boc), Ser(O^(t)Bu),Thr(O^(t)Bu) and Tyr(O^(t)Bu). Two Oxazolidine dipeptides,Fmoc-Gly-Thr(^(ψMe,Me)Pro)-OH and Fmoc-Leu-Ser(^(ψMe,Me)Pro)-OH, wereused in the chain assembly and were obtained from NovaBiochem and usedin the synthesis of the sequence. The protected amino acid derivatives(20 mmol) were dissolved in 100 ml 20% dimethyl sulfoxide (DMSO) in DMF(v/v). Protected amino acids were activated with 20 mM HBTU, 400 mM NMMin 20% DMSO in DMF, and coupling were carried out using two treatmentswith 0.5 mmol protected amino acid, 0.5 mmol HBTU, 1 mmol NMM in 20%DMF/DMSO for 25 minutes and then 40 minutes. Fmoc deprotection reactionswere carried out with two treatments using a 20% piperidine in DMF (v/v)solution for 10 minutes and then 15 minutes. Following synthesis, theresin was then drained, and washed with DCM, DMF, DCM, and then dried invacuo. The peptide-resin was deprotected and released from the resin bytreatment with a TFA/EDT/TIS/H₂O (92.5:2.5:2.5:2.5 (v/v)) solution atroom temperature for 1 hour. The volatiles were then removed with astream of nitrogen gas, the crude peptide precipitated twice with colddiethyl ether and collected by centrifugation. The crude peptide wasthen analyzed on a Waters 2795 analytical RP-HPLC system using a lineargradient (0-60% buffer B in 12 minutes, A: 0.1% TFA in water, B: 0.1%TFA in acetonitrile) on a Jupiter 4 μm Proteo™ 90 Å column. A PE-Sciex™API Electro-spray mass spectrometer was used to confirm correct peptideproduct mass. Crude peptide was obtained in 143 mg yield atapproximately 70% pure based as estimated by analytical RP-HPLCanalysis. Reduced Des-Arg1-ShK (Peptide 1) Retention time (Rt)=5.31minutes, calculated molecular weight=3904.6917 Da (average);Experimental observed molecular weight 3907.0 Da.

Folding of Des-Arq1-ShK (Disulphide bond formation). Following TFAcleavage and peptide precipitation, reduced Des-Arg1-ShK was thenair-oxidized to give the folded peptide. The crude cleaved peptide wasextracted using 20% AcOH in water (v/v) and then diluted with water to aconcentration of approximately 0.15 mg reduced Des-Arg1-ShK per mL, thepH adjusted to about 8.0 using NH₄OH (28-30%), and gently stirred atroom temperature for 36 hours. Folding process was monitored by LC-MSanalysis. Following this, folded Des-Arg1-ShK peptide was purified usingreversed phase HPLC using a 1″ Luna 5 μm C18 100 Å Proteo™ column with alinear gradient 0-40% buffer B in 120 min (A=0.1% TFA in water, B=0.1%TFA in acetonitrile). Folded Des-Arg1-ShK crude peptide eluted earlier(when compared to the elution time in its reduced form) at approximately25% buffer B. Folded Des-Arg1-ShK (Peptide 2) was obtained in 23.2 mgyield in >97% purity as estimated by analytical RP-HPLC analysis (FIG.20A). Calculated molecular weight=3895.7693 Da (monoisotopic),experimental observed molecular weight=3896.5 Da(analyzed on a WatersLCT Premier Micromass MS Technologies). (FIG. 20B). Des-Arg1-ShKdisulfide connectivity was C1-C6, C2-C4, C3-C5.

N-terminal PEGylation of Folded Des-Arq1-ShK. Folded Des-Arg1-ShK,(Peptide 2) was dissolved in water at 1 mg/ml concentration. A 2 MMeO-PEG-Aldehyde, CH₃O—[CH₂CH₂O]n-CH₂CH₂CHO (average molecular weight 20kDa), solution in 50 mM NaOAc, pH 4.5, and a separate 1 M solution ofNaCNBH₃ were freshly prepared. The peptide solution was then added tothe MeO-PEG-Aldehyde containing solution and was followed by theaddition of the NaCNBH₃ solution. The reaction stoichiometry waspeptide:PEG:NaCNBH3 (1:2:0.02), respectively. The reaction was left for48 hours, and was analyzed on an Agilent 1100 RP-HPLC system usingZorbax™ 300SB-C8 5 μm column at 40° C. with a linear gradient (6-60% Bin 16 minutes, A: 0.1% TFA in water, B: 0.1% TFA/90% ACN in water).Mono-pegylated folded Des-Arg1-ShK constituted approximately 58% of thecrude product by analytical RP-HPLC. Mono Pegylated Des-Arg1-ShK wasthen isolated using a HiTrap™ 5 ml SP HP cation exchange column on AKTAFPLC system at 4° C. at 1 mL/min using a gradient of 0-50% B in 25column volumes (Buffers: A=20 mM sodium acetate pH 4.0, B=1 M NaCl, 20mM sodium acetate, pH 4.0). The fractions were analyzed using a 4-20tris-Gly SDS-PAGE gel and RP-HPLC (as described for the crude). SDS-PAGEgels were run for 1.5 hours at 125 V, 35 mA, 5 W. Pooled product wasthen dialyzed at 4° C. in 3 changes of 1 L of A4S buffer (10 mM NaOAc,5% sorbitol, pH 4.0). The dialyzed product was then concentrated in 10 Kmicrocentrifuge filter to 2 mL volume and sterile-filtered using 0.2 μMsyringe filter to give the final product. N-TerminallyPEGylated-Des-Arg1-ShK (Peptide 3) was isolated in 1.7 mg yield with 85%purity as estimated by analytical RP-HPLC analysis (FIG. 23).

The N-Terminally PEGylated-Des-Arg1-ShK, also referred to as“PEG-ShK[2-35]”, was active in blocking human Kv1.3 (FIG. 38A and FIG.38B) as determined by patch clamp electrophysiology (Example 36).

Example 32 N-Terminally PEGylated ShK

The experimental procedures of this working example correspond to theresults shown in FIG. 17.

Peptide Synthesis of reduced ShK. ShK, having the amino acid sequence

(Peptide 4, SEQ ID NO: 5) RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTCwas synthesized in a stepwise manner on a Symphony™ multi-peptidesynthesizer by solid-phase peptide synthesis (SPPS) using2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU)/N-methyl morpholine (NMM)/N,N-dimethyl-formamide (DMF) couplingchemistry at 0.1 mmol equivalent resin scale on Tentagel™-S PHBFmoc-Cys(Trt)-resin. N-alpha-9-fluorenylmethyloxycarbonyl) andside-chain protected amino acids were purchased from Midwest BiotechIncorporated. Fmoc-Cys(Trt)-Tentagel™ resin was purchased from Fluka.The following side-chain protection strategy was employed: Asp(O^(t)Bu),Arg(Pbf), Cys(Trt), Gln(Trt), His(Trt), Lys(N^(ε)-Boc), Ser(O^(t)Bu),Thr(O^(t)Bu) and Tyr(O^(t)Bu). Two Oxazolidine dipeptides,Fmoc-Gly-Thr(^(ψMe,Me)Pro)-OH and Fmoc-Leu-Ser(^(ψMe,Me)Pro)-OH, wereused in the chain assembly and were obtained from NovaBiochem and usedin the synthesis of the sequence. The protected amino acid derivatives(20 mmol) were dissolved in 100 ml 20% dimethyl sulfoxide (DMSO) in DMF(v/v). Protected amino acids were activated with 200 mM HBTU, 400 mM NMMin 20% DMSO in DMF, and coupling were carried out using two treatmentswith 0.5 mmol protected amino acid, 0.5 mmol HBTU, 1 mmol NMM in 20%DMF/DMSO for 25 minutes and then 40 minutes. Fmoc deprotections werecarried out with two treatments using a 20% piperidine in DMF (v/v)solution for 10 minutes and then 15 minutes. Following synthesis, theresin was then drained, and washed with DCM, DMF, DCM, and then dried invacuo. The peptide-resin was deprotected and released from the resin bytreatment with a TFA/EDT/TIS/H₂O (92.5:2.5:2.5:2.5 (v/v)) solution atroom temperature for 1 hour. The volatiles were then removed with astream of nitrogen gas, the crude peptide precipitated twice with colddiethyl ether and collected by centrifugation. The crude peptide wasthen analyzed on a Waters 2795 analytical RP-HPLC system using a lineargradient (0-60% buffer B in 12 minutes, A: 0.1% TFA in water, B: 0.1%TFA in acetonitrile) on a Jupiter 4 μm Proteo™ 90 Å column. A PE-SciexAPI Electro-spray mass spectrometer was used to confirm correct peptideproduct mass. Crude peptide was approximately was obtained 170 mg yieldat about 45% purity as estimated by analytical RP-HPLC analysis. ReducedShK (Peptide 4) Retention time (Rt)=5.054 minutes, calculated molecularweight=4060.8793 Da (average); experimental observed molecularweight=4063.0 Da.

Folding of ShK (Disulphide bond formation). Following TFA cleavage andpeptide precipitation, reduced ShK was then air oxidized to give thefolded peptide. The crude cleaved peptide was extracted using 20% AcOHin water (v/v) and then diluted with water to a concentration ofapproximately 0.15 mg reduced ShK per mL, the pH adjusted to about 8.0using NH₄OH (28-30%), and gently stirred at room temperature for 36hours. Folding process was monitored by LC-MS analysis. Following this,folded ShK peptide was purified by reversed phase HPLC using a 1″ Luna 5μm C18 100 Å Proteo™ column with a linear gradient 0-40% buffer B in 120min (A=0.1% TFA in water, B=0.1% TFA in acetonitrile). Folded ShK crudepeptide eluted earlier (when compared to the elution time in its reducedform) at approximately 25% buffer B. Folded ShK (Peptide 5) was obtainedin 25.5 mg yield in >97% purity as estimated by analytical RP-HPLCanalysis. See FIG. 60. Calculated molecular weight=4051.8764 Da(monoisotopic); experimental observed molecular weight=4052.5 Da(analyzed on Waters LCT Premier

Micromass MS Technologies). ShK disulfide connectivity was C1-C6, C2-C4,and C3-C5.

N-terminal PEGylation of Folded ShK. Folded ShK, having the amino acidsequence

RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC (SEQ ID NO: 5)can be dissolved in water at 1 mg/ml concentration. A 2 MMeO-PEG-Aldehyde, CH₃O—CH₂CH₂O]n-CH₂CH₂CHO (average molecular weight 20kDa), solution in 50 mM NaOAc, pH 4.5 and a separate 1 M solution ofNaCNBH₃ can be freshly prepared. The peptide solution can be then addedto the MeO-PEG-Aldehyde containing solution and can be followed by theaddition of the NaCNBH₃ solution. The reaction stoichiometry can bepeptide:PEG:NaCNBH3 (1:2:0.02), respectively. The reaction can be leftfor 48 hours, and can be analyzed on an Agilent™ 1100 RP-HPLC systemusing Zorbax™ 300SB-C8 5 μm column at 40° C. with a linear gradient(6-60% B in 16 minutes, A: 0.1% TFA in water, B: 0.1% TFA/90% ACN inwater). Mono-pegylated Shk (Peptide 6) can be then isolated using aHiTrap™ 5 mL SP HP cation exchange column on AKTA FPLC system at 4° C.at 1 mL/min using a gradient of 0-50% B in 25 column volumes (Buffers:A=20 mM sodium acetate pH 4.0, B=1 M NaCl, 20 mM sodium acetate, pH4.0). The fractions can be analyzed using a 4-20 tris-Gly SDS-PAGE geland RP-HPLC. SDS-PAGE gels can be run for 1.5 hours at 125 V, 35 mA, 5W. Pooled product can be then dialyzed at 4° C. in 3 changes of 1 L ofA4S buffer (10 mM sodium acetate, 5% sorbitol, pH 4.0). The dialyzedproduct can be then concentrated in 10 K microcentrifuge filter to 2 mLvolume and sterile-filtered using 0.2 μM syringe filter to give thefinal product.

Example 33 N-Terminally PEGylated ShK by Oxime Formation

Peptide Synthesis of reduced ShK. ShK, having the sequence

RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC (SEQ ID NO: 5)can be synthesized in a stepwise manner on a Symphony™ multi-peptidesynthesizer by solid-phase peptide synthesis (SPPS) using2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU)/N-methyl morpholine (NMM)/N,N-dimethyl-formamide (DMF) couplingchemistry at 0.1 mmol equivalent resin scale on Tentagel™-S PHBFmoc-Cys(Trt)-resin. N-alpha-(9-fluorenylmethyloxycarbonyl)- andside-chain protected amino acids can be purchased from Midwest BiotechIncorporated. Fmoc-Cys(Trt)-Tentagel™ resin can be purchased from Fluka.The following side-chain protection strategy can be employed:Asp(O^(t)Bu), Arg(Pbf), Cys(Trt), Gln(Trt), His(Trt), Lys(N^(ε)-Boc),Ser(O^(t)Bu), Thr(O^(t)Bu) and Tyr(O^(t)Bu). Two Oxazolidine dipeptides,Fmoc-Gly-Thr(Ψ^(Me,Me)Pro)-OH and Fmoc-Leu-Ser(ψ^(Me,Me)Pro)-OH, can beused in the chain assembly and can be obtained from NovaBiochem and usedin the synthesis of the sequence. The protected amino acid derivatives(20 mmol) can be dissolved in 100 ml 20% dimethyl sulfoxide (DMSO) inDMF (v/v). Protected amino acids can be activated with 200 mM HBTU, 400mM NMM in 20% DMSO in DMF, and coupling can be carried out using twotreatments with 0.5 mmol protected amino acid, 0.5 mmol HBTU, 1 mmol NMMin 20% DMF/DMSO for 25 minutes and then 40 minutes. Fmoc deprotectionreactions can be carried out with two treatments using a 20% piperidinein DMF (v/v) solution for 10 minutes and then 15 minutes. Following thechain-assembly of the Shk peptide, Boc-amionooxyacetic acid (1.2 equiv)can be coupled at the N-terminus using 0.5 M HBTU in DMF with 4 equivcollidine for 5 minutes. Following synthesis, the resin can be thendrained, and washed with DCM, DMF, DCM, and then dried in vacuo. Thepeptide-resin can be deprotected and released from the resin bytreatment with a TFA/amionooxyacetic acid/TIS/EDT/H2O(90:2.5:2.5:2.5:2.5) solution at room temperature for 1 hour. Thevolatiles can be then removed with a stream of nitrogen gas, the crudepeptide precipitated twice with cold diethyl ether and collected bycentrifugation. The aminooxy-Shk peptide (Peptide 7) can be thenanalyzed on a Waters 2795 analytical RP-HPLC system using a lineargradient (0-60% buffer B in 12 minutes, A: 0.1% TFA in water alsocontaining 0.1% aminooxyacetic acid, B: 0.1% TFA in acetonitrile) on aJupiter 4 μm Proteo™ 90 Å column.

Reversed-Phase HPLC Purification. Preparative Reversed-phasehigh-performance liquid chromatography can be performed on C18, 5 μm,2.2 cm×25 cm) column. Chromatographic separations can be achieved usinglinear gradients of buffer B in A (A=0.1% aqueous TFA; B=90% aq. ACNcontaining 0.09% TFA and 0.1% aminooxyacetic acid), typically 5-95% over90 minutes at 15 mL/min. Preparative HPLC fractions can be characterizedby ESMS and photodiode array (PDA) HPLC, combined and lyophilized.

N-Terminal PEGylation of Shk by Oxime Formation. Lyophilizedaminooxy-Shk (Peptide 7) can be dissolved in 50% HPLC buffer A/B (5mg/mL) and added to a two-fold molar excess of MeO-PEG-Aldehyde,CH₃O—[CH₂CH₂O]_(n)—CH₂CH₂CHO (average molecular weight 20 kDa). Thereaction can be left for 24 hours, and can be analyzed on an Agilent™1100 RP-HPLC system using Zorbax™ 300SB-C8 5 μm column at 40° C. with alinear gradient (6-60% B in 16 minutes, A: 0.1% TFA in water, B: 0.1%TFA/90% ACN in water). Mono-pegylated reduced Shk constitutedapproximately 58% of the crude product by analytical RP-HPLC. MonoPEGylated (oximated) Shk (Peptide 8) can be then isolated using aHiTrap™ 5 mL SP HP cation exchange column on AKTA FPLC system at 4° C.at 1 mL/min using a gradient of 0-50% B in 25 column volumes (Buffers: A20 mM sodium acetate pH 4.0, B=1 M NaCl, 20 mM sodium acetate, pH 4.0).The fractions can be analyzed using a 4-20 tris-Gly SDS-PAGE gel andRP-HPLC. SDS-PAGE gels can be run for 1.5 hours at 125 V, 35 mA, 5 W.Pooled product can be then dialyzed at 4° C. in 3 changes of 1 L of A4Sbuffer (10 mM NaOAc, 5% sorbitol, pH 4.0). The dialyzed product can bethen concentrated in 10 K microcentrifuge filter to 2 mL volume andsterile-filtered using 0.2 μM syringe filter to give the final product.

Folding of ShK (Disulphide bond formation). The mono-PEGylated(oximated) Shk can be dissolved in 20% AcOH in water (v/v) and can bethen diluted with water to a concentration of approximately 0.15 mgpeptide mL, the pH adjusted to about 8.0 using NH₄OH (28-30%), andgently stirred at room temperature for 36 hours. Folding process can bemonitored by LC-MS analysis. Following this, folded mono-PEGylated(oximated) Shk (Peptide 9) can be purified using by reversed phase HPLCusing a 1′ Luna 5 μm C18 100 Å Proteo™ column with a linear gradient0-40% buffer B in 120 min (A=0.1% TFA in water, B=0.1% TFA inacetonitrile). Mono-PEGylated (oximated) ShK disulfide connectivity canbe C1-C6, C2-C4, and C3-C5.

Example 34 N-Terminally PEGylated ShK (Amidation)

The experimental procedures of this working example correspond to theresults shown in FIG. 18.

N-Terminal PEGylation of Shk by Amide Formation. A 10 mg/mL solution offolded Shk (Peptide 5), in 100 mM Bicine pH 8.0, can be added to solidsuccinimidyl ester of 20 kDa PEG propionic acid (mPEG-SPA;CH₃O—[CH₂CH₂O]n-CH₂CH₂CO—NHS) at room temperature using a 1.5 molarexcess of the mPEG-SPA to Shk. After one hour with gentle stirring, themixture can be diluted to 2 mg/mL with water, and the pH can be adjustedto 4.0 with dilute HCl. The extent of mono-pegylated Shk (Peptide 10),some di-PEGylated Shk or tri-PEGylated Shk, unmodified Shk andsuccinimidyl ester hydrolysis can be determined by SEC HPLC using aSuperdex™ 75 HR 10/30 column (Amersham) eluted with 0.05 M NaH₂PO₄, 0.05M Na₂HPO₄, 0.15 M NaCl, 0.01 M NaN₃, pH 6.8, at 1 mL/min. The fractionscan be analyzed using a 4-20 tris-Gly SDS-PAGE gel and RP-HPLC. SDS-PAGEgels can be run for 1.5 hours at 125 V, 35 mA, 5 W. Pooled product canbe then dialyzed at 4° C. in 3 changes of 1 L of A4S buffer (10 mMNaOAc, 5% sorbitol, pH 4.0). The dialyzed N-terminally PEGylated(amidated) ShK (Peptide 10) can be then concentrated in 10 Kmicrocentrifuge filter to 2 mL volume and sterile-filtered using 0.2 μMsyringe filter to give the final product.

Example 35 Fc-L-SmIIIA

Fc-SmIIIA expression vector. A 104 bp BamHI-NotI fragment containingpartial linker sequence and SmIIIA peptide encoded with human highfrequency codons was assembled by PCR with overlapping primers 3654-50and 3654-51 and cloned into to the 7.1 kb NotI-BamHI back bone togenerate pcDNA3.1(+) CMVi-hFc-SmIIIA as described in Example 1.

  BamHI 5′GGATCCGGAGGAGGAGGAAGCTGCTGCAACGGCCGCCGCGGCTGCAGCAGCCGCTGG                       C  C  N  G  R  R  G  C  S  S  R  WTGCCGCGACCACAGCCGCTGCTGCTGAGCGGCCGC3′ //SEQ ID NO: 872C  R  D  H  S  R  C  C     NotI SEQ ID NO: 873 Forward 5′-3′:GGAGGAGGATCCGGAGGAGGAGGAAGCTGCTGCAACGGCCGCCGCGGCTGCAGCAGC CGC //SEQ ID NO: 874Reverse 5′-3′:ATTATTGCGGCCGCTCAGCAGCAGCGGCTGTGGTCGCGGCACCAGCGGCTGCTGCAG CCGC SEQ ID NO: 875The sequences of the BamHI to NotI fragments in the final constructswere verified by sequencing.

Transient expression of Fc-L-SmIIIa. 7.5 ug of the toxin peptide Fcfusion construct pcDNA3.1(+) CMVi-hFc-SmIIIA were transfected into 293-Tcells in 10 cm tissue culture plate with FuGENE 6 as transfectionreagent. Culture medium was replaced with serum-free medium at 24 hourspost-transfection and the conditioned medium was harvested at day 5post-transfection. Transient expression of Fc-SmIIIA from 293-T cellswas analyzed by Western blot probed with anti-hFc antibody (FIG. 25A andFIG. 25B). Single band of expressed protein with estimated MW was shownin both reduced and non-reduced samples. Transient expression level ofFc-SmIIIA was further determined to be 73.4 μg/ml according to ELISA.

Example 36 Electrophysiology Experiments

Cell Culture. Stable cell line expressing human Kv1.3 channel waslicensed from Biofocus. Cells were kept at 37° C. in 5% CO₂ environment.Culture medium contains DMEM with GlutaMax™ (Invitrogen), 1×non-essential amino acid, 10% fetal bovine serum and 500 μg/mLgeneticin. Cells were plated and grown at low confluence on 35 mmculture dishes for at least 24 hours prior to electrophysiologyexperiments.

Electrophysiology Recording by Patch Clamping. Whole-cell currents wererecorded from single cells by using tight seal configuration of thepatch-clamp technique. A 35 mm culture dish was transferred to therecording stage after rinsing and replacing the culture medium withrecording buffer containing 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 10 mMHEPES, and 5 mM Glucose. pH was adjusted to 7.4 with NaOH and theosmolarity was set at 300 mOsm. Cells were perfused continuously withthe recording buffer via one of the glass capillaries arranged inparallel and attached to a motorized rod, which places the glasscapillary directly on top of the cell being recorded. Recording pipettesolution contained 90 mM K-gluconate, 20 mM KF, 10 mM NaCl, 1 mMMgCl₂-6H₂O, 10 mM EGTA, 5 mM K₂-ATP, and 10 mM HEPES. The pH for theinternal solution was adjusted to 7.4 with KOH and the osmolarity wasset at 280 mOsm. Experiments were performed at room temperature (20-22°C.) and recorded using Multiclamp™ 700 A amplifier (Molecular DevicesInc.). Pipette resistances were typically 2-3 MΩ.

Protein toxin potency determination on Kv1.3 current: HEK293 cellsstably expressing human Kv1.3 channel were voltage clamped at −80 mVholding potential. Outward Kv1.3 currents were activated by giving 200msec long depolarizing steps to +30 mV from the holding potential of −80mV and filtered at 3 kHz. Each depolarizing step was separated from thesubsequent one with a 10 s interval. Analogue signals were digitized byDigidata™ 1322A digitizer (Molecular Devices) subsequently stored oncomputer disk for offline analyses using Clampfit™ 9 (Molecular DevicesInc.). In all studies, stable baseline Kv1.3 current amplitudes wereestablished for 4 minutes before starting the perfusion of the proteintoxin at incremental concentrations. A steady state block was alwaysachieved before starting the perfusion of the subsequent concentrationof the protein toxin.

Data analysis. Percent of control (POC) is calculated based on thefollowing equation: (Kv1.3 current after protein toxin addition/Kv1.3current in control)*100. At least 5 concentrations of the protein toxin(e.g. 0.003, 0.01, 0.03, 0.1, 0.3, 100 nM) were used to calculate theIC₅₀ value. IC₅₀ values and curve fits were estimated using the fourparameter logistic fit of XLfit software (Microsoft Corp.). IC₅₀ valuesare presented as mean value±s.e.m. (standard error of the mean).

Drug preparations. Protein toxins (typically 10-100 μM) were dissolvedin distilled water and kept frozen at −80° C. Serial dilutions of thestock protein toxins were mixed into the recording buffer containing0.1% bovine serum albumin (BSA) and subsequently transferred to glassperfusion reservoirs. Electronic pinch valves controlled the flow of theprotein toxin from the reservoirs onto the cell being recorded.

Example 37 Immunobiology and Channel Binding

Inhibition of T cell cytokine Production following PMA and anti-CD3antibody stimulation of PBMCs. PBMC's were previously isolated fromnormal human donor Leukophoresis packs, purified by density gradientcentrifugation (Ficoll Hypaque), cryopreserved in CPZ CryopreservationMedium Complete (INCELL, MCPZF-100 plus 10% DMSO final). PBMC's werethawed (95% viability), washed, and seeded at 2×10⁵ cells per well inculture medium (RPMI medium 1640; GIBCO) supplemented with 10% fetalcalf serum, 100 U/ml penicillin, 100 mg/ml streptomycin 2 mML-glutamine, 100 uM non-essential amino acids, and 20 uM 2-ME) in96-well flat-bottom tissue culture plates. Cells were pre-incubated withserially diluted (100 nM-0.001 nM final) ShK[1-35], Fc-L10-ShK[1-35] orfc control for 90 min before stimulating for 48 hr with PMA/anti-CD3 (1ng/ml and 50 ng/ml, respectively) in a final assay volume of 200 ul.Analysis of the assay samples was performed using the Meso ScaleDiscovery (MSD) SECTOR™ Imager 6000 (Meso Scale Discovery, Gaithersbury,Md.) to measure the IL-2 and IFNg protein levels by utilizingelectrochemiluminescence (ECL). The conditioned medium (50 ul) was addedto the MSD Multi-spot 96-well plates (each well containing three captureantibodies; IL-2, TNF, IFNγ). The plates were sealed, wrapped in tinfoil, and incubated at room temperature on a plate shaker for 2 hr. Thewells were washed 1× with 200 ul PBST (BIOTEK, Elx405 Auto PlateWasher). For each well, 20 ul of Ruthenium-labeled detection antibodies(1 μg/ml final in Antibody Dilution Buffer; IL-1, TNF, IFNγ) and 130 ulof 2×MSD Read Buffer added, final volume 150 ul. The plates were sealed,wrapped in tin foil, and incubated at room temperature on a plate shakerfor 1 hr. The plates were then read on the SECTOR™ Imager 6000. FIGS.35A & 35B shows the CHO-derived Fc-L10-ShK[1-35] peptibody potentlyinhibits IL-2 and IFNg production from T cells in a dose-dependentmanner. Compared to native ShK[1-35] peptide, the peptibody produces agreater extent of inhibition (POC=Percent Of Control of response in theabsence of inhibitor).

Inhibition of T cell cytokine production following anti-CD3 andanti-CD28 antibody stimulation of PBMCs. PBMCs were previously isolatedfrom normal human donor Leukopheresis packs, purified by densitygradient centrifugation (Ficoll Hypaque), and cryopreserved using INCELLFreezing Medium. PBMCs were thawed (95% viability), washed, and seeded(in RPMI complete medium containing serum replacement, PSG) at 2×10⁵cells per well into 96-well flat bottom plates. Cells were pre-incubatedwith serially diluted (100 nM-0.003 nM final) ShK[1-35],Fc-L10-ShK[1-35], or Fc control for 1 hour before the addition of aCD3and aCD28 (2.5 ng/mL and 100 ng/mL respectively) in a final assay volumeof 200 mL. Supernatants were collected after 48 hours, and analyzedusing the Meso Scale Discovery (MSD) SECTOR™ Imager 6000 (Meso ScaleDiscovery, Gaithersbury, Md.) to measure the IL-2 and IFNg proteinlevels by utilizing electrochemiluminescence (ECL). 20 mL of supernatantwas added to the MSD multi-spot 96-well plates (each well containingIL-2, TNFa, and IFNg capture antibodies). The plates were sealed andincubated at room temperature on a plate shaker for 1 hour. Then 20 mLof Ruthenium-labeled detection antibodies (1 μg/ml final of IL-2, TNFα,and IFNγ in Antibody Dilution Buffer) and 110 mL of 2×MSD Read Bufferwere added. The plates were sealed, covered with tin foil, and incubatedat room temperature on a plate shaker for 1 hour. The plates were thenread on the SECTOR™ Imager 6000. FIGS. 37A & 37B shows the CHO-derivedFc-L10-ShK[1-35] peptibody potently inhibits IL-2 and IFNg productionfrom T cells in a dose-dependent manner. Compared to native ShK[1-35]peptide which shows only partial inhibition, the peptibody producesnearly complete inhibition of the inflammatory cytokine response.(POC=Percent Of Control of response in the absence of inhibitor).

Inhibition of T cell proliferation following anti-CD3 and anti-CD28antibody stimulation of PBMCs. PBMC's were previously isolated fromnormal human donor Leukophoresis packs, purified by density gradientcentrifugation (Ficoll Hypaque), cryopreserved in CPZ CryopreservationMedium Complete (INCELL, MCPZF-100 plus 10% DMSO final). PBMC's werethawed (95% viability), washed, and seeded at 2×10⁵ cells per well inculture medium (RPMI medium 1640; GIBCO) supplemented with 10% fetalcalf serum, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mML-glutamine, 100 μM non-essential amino acids, and 20 μM 2-ME) in96-well flat-bottom tissue culture plates. Cells were pre-incubated witheither anti-human CD32 (FcyRII) blocking antibody (per manufacturersinstructions EASY SEP Human Biotin Selection Kit #18553, StemCellTechnologies Vancouver, BC) or Fc-L10-ShK (100 nM-0.001 nM final) for 45min. Fc-L10-ShK (100 nM-0.001 nM final) was then added to the cellscontaining anti-human CD32 blocking antibody while medium was added tothe cells containing Fc-L10-ShK. Both sets were incubated for anadditional 45 min before stimulating for 48 hr with aCD3/aCD28 (0.2ng/ml and 100 ng/ml, respectively). Final assay volume was 200 ul.[3H]TdR (1 uCi per well) was added and the plates were incubated for anadditional 16 hrs. Cells were then harvested onto glass fiber filtersand radioactivity was measured in a B-scintillation counter. FIGS. 36A &36B shows the CHO-derived Fc-L10-ShK[1-35] peptibody potently inhibitsproliferation of T cells in a dose-dependent manner. Pre-blocking withthe anti-CD32 (FcR) blocking antibody has little effect on thepeptibodies ability to inhibit T cell proliferation suggesting Kv1.3inhibition and not FcR binding is the mechanism for the inhibitionobserved (POC=Percent Of Control of response in the absence ofinhibitor).

Immunohistochemistry analysis of Fc-L10-ShK[1-35] binding to HEK 293cells overexpressing human Kv1.3. HEK 293 cells overexpressing humanKv1.3 (HEK Kv1.3) were obtained from BioFocus plc (Cambridge, UK) andmaintained per manufacturer's recommendation. The parental HEK 293 cellline was used as a control. Cells were plated on Poly-D-Lysine 24 wellplates (#35-4414; Becton-Dickinson, Bedford, Mass.) and allowed to growto approximately 70% confluence. HEK KV1.3 were plated at 0.5×10e5cells/well in 1 ml/well of medium. HEK 293 cells were plated at adensity of 1.5×10e5 cells/well in 1 ml/well of medium. Before staining,cells were fixed with formalin (Sigma HT50-1-1 Formalin solution,diluted 1:1 with PBS/0.5% BSA before use) by removing cell growthmedium, adding 0.2 ml/well formalin solution and incubating at roomtemperature for ten minutes. Cells were stained by incubating with 0.2ml/well of 5 μg/ml Fc-L10-ShK[1-35] in PBS/BSA for 30′ at roomtemperature. Fc-L10-ShK[1-35] was aspirated and then the cells werewashed one time with PBS/0.5% BSA. Detection antibody (Goat F(ab)₂anti-human IgG-phycoerythrin; Southern Biotech Associates, Birmingham,Ala.) was added to the wells at 5 μg/ml in PBS/0.5% BSA and incubatedfor 30′ at room temperature. Wash cells once with PBS/0.5% BSA andexamine using confocal microscopy (LSM 510 Meta Confocal Microscope;Carl Zeiss AG, Germany). FIG. 33B shows the Fc-L10-ShK[1-35] peptibodyretains binds to Kv1.3 overexpressing HEK 293 cells but shows littlebinding to untransfected cells (FIG. 33A) indicating theFc-L10-ShK[1-35] peptibody can be used as a reagent to detect cellsoverexpressing the Kv1.3 channel. In disease settings where activated Teffector memory cells have been reported to overproduce Kv1.3, thisreagent can find utility in both targeting these cells and in theirdetection.

An ELISA assay demonstrating Fc-L10-ShK[1-35] binding to fixed HEK 293cells overexpressing Kv1.3. FIG. 34A shows a dose-dependent increase inthe peptibody binding to fixed cells that overexpress Kv1.3,demonstrating that the peptibody shows high affinity binding to itstarget and the utility of the Fc-L10-ShK[1-35] molecule in detection ofcells expressing the channel. Antigen specific T cells that causedisease in patients with multiple sclerosis have been shown tooverexpress Kv1.3 by whole cell patch clamp electrophysiology,—alaborius approach. Our peptibody reagent can be a useful and convenienttool for monitoring Kv1.3 channel expression in patients and haveutility in diagnostic applications. The procedure shown in FIG. 34A andFIG. 34B follows.

FIG. 34A. A whole cell immunoassay was performed to show binding ofintact Fc-L10-ShK[1-35] to Kv1.3 transfected HEK 293 cells (BioFocusplc, Cambridge, UK). Parent HEK 293 cells or HEK Kv1.3 cells were platedat 3×10e4 cells/well in poly-D-Lysine coated ninety-six well plates(#35-4461; Becton-Dickinson, Bedford, Mass.). Cells were fixed withformalin (Sigma HT50-1-1 Formalin solution, diluted 1:1 with PBS/0.5%BSA before use) by removing cell growth medium, adding 0.2 ml/wellformalin solution and incubating at room temperature for 25 minutes andthen washing one time with 100 μl/well of PBS/0.5% BSA. Wells wereblocked by addition of 0.3 ml/well of BSA blocker (50-61-00; KPL 10% BSADiluent/Blocking Solution, diluted 1:1 with PBS; KPL, Gaithersburg, Md.)followed by incubation at room temperature, with shaking, for 3 hr.Plates were washed 2 times with 1×KP Wash Buffer (50-63-00; KPL).Samples were diluted in Dilution Buffer (PBS/0.5% Tween-20) or DilutionBuffer with 1% Male Lewis Rat Serum (RATSRM-M; Bioreclamation Inc.,Hicksville, N.Y.) and 0.1 ml/well was added to blocked plates,incubating for 1 hr at room temperature with shaking. Plates were washed3 times with 1xKP Wash Buffer and then incubated with HRP-Goatanti-human IgG Fc (#31416; Pierce, Rockford, Ill.) diluted 1:5000 inPBS/0.1% Tween-20 for 1 hr at room temperature, with shaking. Plateswere washed plates 3 times with 1xKP Wash Buffer, and then 0.1 ml/wellTMB substrate (52-00-01; KPL) was added. The reactions were stopped byaddition of 0.1 ml/well 2 N Sulfuric Acid. Absorbance was read at 450 nmon a Molecular Devices SpectroMax 340 (Sunnyvale, Calif.).

FIG. 34B. Whole cell immunoassay was performed as above with thefollowing modifications. HEK 293 cells were plated at 1×10e5 cells/welland HEK Kv1.3 cells were plated at 6×10e4 cells/well in poly-D-Lysinecoated 96 well plates. Fc Control was added at 500 ng/ml in a volume of0.05 ml/well. HRP-Goat anti-human IgG Fc (#31416; Pierce, Rockford,Ill.) was diluted 1:10,000 in PBS/0.1% Tween-20. ABTS (50-66-00, KPL)was used as the substrate. Absorbances were read at 405 nm afterstopping reactions by addition of 0.1 ml/well of 1% SDS.

Example 38 Purification of Fc-L10-ShK(1-35)

Expression of Fc-L10-ShK[1-35] was as described in Example 3 hereinabove. Frozen, E. coli paste (18 g) was combined with 200 ml of roomtemperature 50 mM tris HCl, 5 mM EDTA, pH 8.0 and was brought to about0.1 mg/ml hen egg white lysozyme. The suspended paste was passed througha chilled microfluidizer twice at 12,000 PSI. The cell lysate was thencentrifuged at 22,000 g for 15 min at 4° C. The pellet was thenresuspended in 200 ml 1% deoxycholic acid using a tissue grinder andthen centrifuged at 22,000 g for 15 min at 4° C. The pellet was thenresuspended in 200 ml water using a tissue grinder and then centrifugedat 22,000 g for 15 min at 4° C. The pellet (3.2 g) was then dissolved in32 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. The pellet solution wasthen centrifuged at 27,000 g for 15 min at room temperature, and then 5ml of the supernatant was transferred to 500 ml of the refolding buffer(3 M urea, 20% glycerol, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1mM cystamine HCl, 4 mM cysteine, pH 9.5) at 4° C. with vigorousstirring. The stirring rate was then slowed and the incubation wascontinued for 2 days at 4° C. The refolding solution was then stored at−70° C.

The stored refold was defrosted and then diluted with 2 L of water andthe pH was adjusted to 7.3 using 1 M H₃PO₄. The pH adjusted material wasthen filtered through a 0.22 μm cellulose acetate filter and loaded onto a 60 ml Amersham SP-FF (2.6 cm I.D.) column at 20 ml/min in S-BufferA (20 mM NaH2PO4, pH 7.3) at 7° C. The column was then washed withseveral column volumes of S-Buffer A, followed by elution with a lineargradient from 0% to 60% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.3)followed by a step to 100% S-Buffer B at 10 ml/min 7° C. Fractions werethen analyzed using a Coomassie brilliant blue stained tris-glycine4-20% SDS-PAGE, and the fractions containing the desired product werepooled based on these data. The pool was then loaded on to a 1 mlAmersham rProtein A HiTrap column in PBS at 1 ml/min 7° C. Then columnwas then washed with several column volumes of 20 mM NaH₂PO₄ pH 6.5, 1 MNaCl and eluted with 100 mM glycine pH 3.0. To the elution peak, 0.0125volumes (25 ml) of 3 M sodium acetate was added.

A spectral scan was then conducted on 50 μl of the combined pool dilutedin 700 μl water using a Hewlett Packard 8453 spectrophotometer (FIG.46A). The concentration of the filtered material was determined to be2.56 mg/ml using a calculated molecular mass of 30,410 g/mol andextinction coefficient of 36,900 M−1 cm−1. The purity of the filteredmaterial was then assessed using a Coomassie brilliant blue stainedtris-glycine 4-20% SDS-PAGE (FIG. 46B). The macromolecular state of theproduct was then determined using size exclusion chromatography on 20 μgof the product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm (FIG. 46C). The product was then subject tomass spectral analysis by diluting 1 μl of the sample into 10 μl ofsinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50%acetonitrile). One milliliter of the resultant solution was spotted ontoa MALDI sample plate. The sample was allowed to dry before beinganalyzed using a Voyager DE-RP time-of-flight mass spectrometer equippedwith a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear modewas used, with an accelerating voltage of 25 kV. Each spectrum wasproduced by accumulating data from ˜200 laser shots. External masscalibration was accomplished using purified proteins of known molecularmasses. The product was then stored at −80° C.

The IC₅₀ for blockade of human Kv1.3 by purified E. coli-derivedFc-L10-ShK[1-35], also referred to as “Fc-L-ShK[1-35]”, is shown inTable 35 (in Example 50 herein below).

Example 39 Purification of Bacterially Expressed Fc-L10-ShK(2-35)

Expression of Fc-L10-ShK[2-35] was as described in Example 4 hereinabove. Frozen, E. coli paste (16.5 g) was combined with 200 ml of roomtemperature 50 mM tris HCl, 5 mM EDTA, pH 8.0 and was brought to about0.1 mg/ml hen egg white lysozyme. The suspended paste was passed througha chilled microfluidizer twice at 12,000 PSI. The cell lysate was thencentrifuged at 22,000 g for 15 min at 4° C. The pellet was thenresuspended in 200 ml 1% deoxycholic acid using a tissue grinder andthen centrifuged at 22,000 g for 15 min at 4° C. The pellet was thenresuspended in 200 ml water using a tissue grinder and then centrifugedat 22,000 g for 15 min at 4° C. The pellet (3.9 g) was then dissolved in39 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. The pellet solution wasthen centrifuged at 27,000 g for 15 min at room temperature, and then 5ml of the supernatant was transferred to 500 ml of the refolding buffer(3 M urea, 20% glycerol, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1mM cystamine HCl, 4 mM cysteine, pH 9.5) at 4° C. with vigorousstirring. The stirring rate was then slowed and the incubation wascontinued for 2 days at 4° C. The refolding solution was then stored at−70° C.

The stored refold was defrosted and then diluted with 2 L of water andthe pH was adjusted to 7.3 using 1 M H₃PO₄. The pH adjusted material wasthen filtered through a 0.22 μm cellulose acetate filter and loaded onto a 60 ml Amersham SP-FF (2.6 cm I.D.) column at 20 ml/min in S-BufferA (20 mM NaH₂PO₄, pH 7.3) at 7° C. The column was then washed withseveral column volumes of S-Buffer A, followed by elution with a lineargradient from 0% to 60% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.3)followed by a step to 100% S-Buffer B at 10 ml/min 7° C. The fractionscontaining the desired product were pooled and filtered through a 0.22μm cellulose acetate filter. The pool was then loaded on to a 1 mlAmersham rProtein A HiTrap column in PBS at 2 ml/min 7° C. Then columnwas then washed with several column volumes of 20 mM NaH₂PO₄ pH 6.5, 1 MNaCl and eluted with 100 mM glycine pH 3.0. To the elution peak, 0.0125volumes (18 ml) of 3 M sodium acetate was added, and the sample wasfiltered through a 0.22 μm cellulose acetate filter.

A spectral scan was then conducted on 20 μl of the combined pool dilutedin 700 μl water using a Hewlett Packard 8453 spectrophotometer (FIG.40A). The concentration of the filtered material was determined to be3.20 mg/ml using a calculated molecular mass of 29,282 g/mol andextinction coefficient of 36,900 M⁻¹ cm⁻¹. The purity of the filteredmaterial was then assessed using a Coomassie brilliant blue stainedtris-glycine 4-20% SDS-PAGE (FIG. 40B). The macromolecular state of theproduct was then determined using size exclusion chromatography on 50 μgof the product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm (FIG. 40C). The product was then subject tomass spectral analysis by diluting 1 μl of the sample into 10 μl ofsinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50%acetonitrile). One milliliter of the resultant solution was spotted ontoa MALDI sample plate. The sample was allowed to dry before beinganalyzed using a Voyager DE-RP time-of-flight mass spectrometer equippedwith a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear modewas used, with an accelerating voltage of 25 kV. Each spectrum wasproduced by accumulating data from ˜200 laser shots. External masscalibration was accomplished using purified proteins of known molecularmasses (FIG. 40D). The product was then stored at −80° C.

The IC₅₀ for blockade of human Kv1.3 by purified E. coli-derivedFc-L10-ShK[2-35], also referred to as “Fc-L-ShK[2-35]”, is shown inTable 35 (in Example 50 herein below).

Example 40 Purification of Bacterially Expressed Fc-L10-OsK1

Frozen, E. coli paste (129 g; see Example 10) was combined with 1290 mlof room temperature 50 mM tris HCl, 5 mM EDTA, pH 7.8 and was brought toabout 0.1 mg/ml hen egg white lysozyme. The suspended paste was passedthrough a chilled microfluidizer twice at 12,000 PSI. The cell lysatewas then centrifuged at 17,700 g for 15 min at 4° C. The pellet was thenresuspended in 1290 ml 1% deoxycholic acid using a tissue grinder andthen centrifuged at 17,700 g for 15 min at 4° C. The pellet was thenresuspended in 1290 ml water using a tissue grinder and then centrifugedat 17,700 g for 15 min at 4° C. 8 g of the pellet (16.3 g total) wasthen dissolved in 160 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. 100ml of the pellet solution was then incubated with 1 ml of 1 M DTT for 60min at 37° C. The reduced material was transferred to 5000 ml of therefolding buffer (1 M urea, 50 mM tris, 160 mM arginine HCl, 2.5 mMEDTA, 1.2 mM cystamine HCl, 4 mM cysteine, pH 10.5) at 2 ml/min, 4° C.with vigorous stirring. The stirring rate was then slowed and theincubation was continued for 3 days at 4° C.

The pH of the refold was adjusted to 8.0 using acetic acid. The pHadjusted material was then filtered through a 0.22 μm cellulose acetatefilter and loaded on to a 50 ml Amersham Q Sepharose-FF (2.6 cm I.D.)column at 10 ml/min in Q-Buffer A (20 mM Tris, pH 8.5) at 8° C. with aninline 50 Amersham Protein A column (2.6 cm I.D.). After loading, the QSepharose column was removed from the circuit, and the remainingchromatography was carried out on the protein A column. The column waswashed with several column volumes of Q-Buffer A, followed by elutionusing a step to 100 mM glycine pH 3.0. The fractions containing thedesired product were pooled and immediately loaded on to a 50 mlAmersham SP-Sepharose HP column (2.6 cm I.D.) at 20 ml/min in S-Buffer A(20 mM NaH₂PO₄, pH 7.0) at 8° C. The column was then washed with severalcolumn volumes of S-Buffer A followed by a linear gradient from 5% to60% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.0) followed by a step to100% S-Buffer B. Fractions were then analyzed using a Coomassiebrilliant blue stained tris-glycine 4-20% SDS-PAGE. The fractionscontaining the bulk of the desired product were pooled and then appliedto a 75 ml MEP Hypercel column (2.6 cm I.D.) at 5 ml/min in MEP Buffer A(20 mM tris, 200 mM NaCl, pH 8.0) at 8° C. Column was eluted with alinear gradient from 5% to 50% MEP Buffer B (50 mM sodium citrate pH4.0) followed by a step to 100% MEP Buffer B. Fractions were thenanalyzed using a Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE, and the fractions containing the bulk of the desired productwere pooled.

The MEP pool was then concentrated to about 20 ml using a Pall Jumbo-Sepwith a 10 kDa membrane followed by buffer exchange with FormulationBuffer (20 mM NaH₂PO₄, 200 mM NaCl, pH 7.0) using the same membrane. Aspectral scan was then conducted on 50 μl of the combined pool dilutedin 700 μl Formulation Buffer using a Hewlett Packard 8453spectrophotometer (FIG. 41A). The concentration of the material wasdetermined to be 4.12 mg/ml using a calculated molecular mass of 30,558g/mol and extinction coefficient of 35,720 M⁻¹ cm⁻¹. The purity of thematerial was then assessed using a Coomassie brilliant blue stainedtris-glycine 4-20% SDS-PAGE (FIG. 41B). The macromolecular state of theproduct was then determined using size exclusion chromatography on 123μg of the product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm (FIG. 41C). The product was then subject tomass spectral analysis by chromatographing approximately 4 μg of thesample through a RP-HPLC column (Vydac C₄, 1×150 mm). Solvent A was 0.1%trifluoroacetic acid in water and solvent B was 0.1% trifluoroaceticacid in 90% acetonitrile, 10% water. The column was pre-equilibrated in10% solvent B at a flow rate of 80 μl per min. The protein was elutedusing a linear gradient of 10% to 90% solvent B over 30 min. Part of theeffluent was directed into a LCQ ion trap mass spectrometer. The massspectrum was deconvoluted using the Bioworks software provided by themass spectrometer manufacturer. (FIG. 41D). The product was filteredthrough a 0.22 μm cellulose acetate filter and then stored at −80° C.

The yield for the E. coli-expressed Fc-L10-OSK1 prep was 81 mg from 40 gof cell paste (129 g×(8 g/16.3 g)×(100 ml/160 ml)=39.6 g which wasrounded to 40 g), the purity was greater than 80% judging by SDS-PAGE,it is running as the expected dimer judging by SEC-HPLC, and the masswas within the expected molecular weight range judging by MS.

The IC₅₀ for blockade of human Kv1.3 by purified E. coli-derivedFc-L10-OSK1, also referred to as “Fc-L-OSK1”, is shown in Table 35 (inExample 50 herein below).

Example 41 Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], andFc-L10-OSK1 [K7S,E16K,K20D] Expressed by Mammalian Cells

Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D], inhibitors of Kv1.3, were expressed in mammalian cells.A DNA sequence coding for the Fc region of human IgG1 fused in-frame toa linker sequence and a monomer of the Kv1.3 inhibitor peptide OSK1,OSK1[K7S], OSK1[E16K,K20D], or OSK1[K7S,E16K,K20D] was constructed asdescribed below. Methods for expressing and purifying the peptibody frommammalian cells (HEK 293 and Chinese Hamster Ovary cells) are disclosedherein.

For construction of Fc-L10-OSK1, Fc-L10-OSK1[K7S],Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] expressionvectors, a PCR strategy was employed to generate the full length genes,OSK1, OSK1[K7S], OSK1[E16K,K20D], and OSK1[K7S,E16K,K20D], each linkedto a four glycine and one serine amino acid linker with two stop codonsand flanked by BamHI and NotI restriction sites as shown below.

Two oligos for each of OSK1, OSK1[K7S], OSK1[E16K,K20D], and[K7S,E16K,K20D]OSK1 with the sequence as depicted below were used in aPCR reaction with PfuTurbo HotStart DNA polymerase (Stratagene) at 95°C.-30 sec, 55° C.-30 sec, 75° C.-45 sec for 35 cycles; BamHI (ggatcc)and NotI (gcggccgc) restriction sites are underlined.

OSK1: Forward primer: cat gga tcc gga gga gga (SEQ IDgga agc ggc gtg atc atc aac gtg aag tgc NO: 876)aag atc agc cgc cag tgc ctg gag ccc tgc aag aag gcc g;Reverse primer: cat gcg gcc gct tac tac (SEQ IDttg ggg gtg cag tgg cac ttg ccg ttc atg NO: 877)cac ttg ccg aag cgc atg ccg gcc ttc ttg cag ggc tcc a; OSK1[K7S]:Forward primer: cat gga tcc gga gga gga (SEQ IDgga agc ggc gtg atc atc aac gtg agc tgc NO: 878)aag atc agc cgc cag tgc ctg gag ccc tgc aag aag gcc g;Reverse primer: cat gcg gcc gct tac tac (SEQ IDttg ggg gtg cag tgg cac ttg ccg ttc atg NO: 879)cac ttg ccg aag cgc atg ccg gcc ttc ttg cag ggc tcc a; OSK1[E16K, K20D]:Forward primer: cat gga tcc gga gga gga (SEQ IDgga agc ggc gtg atc atc aac gtg aag tgc NO: 880)aag atc agc cgc cag tgc ctg aag ccc tgc aag gac gcc g;Reverse primer: cat gcg gcc gct tac tac (SEQ IDttg ggg gtg cag tgg cac ttg ccg ttc atg NO: 881)cac ttg ccg aag cgc atg ccg gcg tcc ttg cag ggc ttc a;OSK1[K7S, E16K, K20D]: Forward primer: cat gga tcc gga gga gga (SEQ IDgga agc ggc gtg atc atc aac gtg agc tgc NO: 882)aag atc agc cgc cag tgc ctg aag ccc tgc aag gac gcc g;Reverse primer: cat gcg gcc gct tac tac (SEQ IDttg ggg gtg cag tgg cac ttg ccg ttc atg NO: 883)cac ttg ccg aag cgc atg ccg gcg tcc ttg cag ggc ttc a.

The resulting PCR products were resolved as the 155 bp bands on a fourpercent agarose gel. The 155 bp PCR product was purified using PCRPurification Kit (Qiagen), then digested with BamHI and NotI (Roche)restriction enzymes, and agarose gel was purified by Gel Extraction Kit(Qiagen). At the same time, the pcDNA3.1(+) CMVi-hFc-Shk[2-35] vectorwas digested with BamHI and NotI restriction enzymes and the largefragment was purified by Gel Extraction Kit. The gel purified PCRfragment was ligated to the purified large fragment and transformed intoOne Shot® Top10F′ (Invitrogen). DNAs from transformed bacterial colonieswere isolated and digested with BamHI and NotI restriction enzymes andresolved on a two percent agarose gel. DNAs resulting in an expectedpattern were submitted for sequencing. Although, analysis of severalsequences of clones yielded a 100% percent match with the abovesequences, only one clone from each gene was selected for large scaledplasmid purification. The DNA of Fc-L10-OSK1, Fc-L10-OSK1[K7S],Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] in pCMVi vectorwas resequenced to confirm the Fc and linker regions and the sequencewas 100% identical to the above sequences. The sequences and pictorialrepresentations of Fc-L10-OSK1, Fc-L10-OSK1[K7S],Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] are depicted inFIG. 42A-B, FIG. 43A-B, FIG. 44A-B and FIG. 45A-B, respectively.

HEK-293 cells used in transient transfection expression of Fc-L110-OSK1,Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D]in pCMVi protein were cultured in growth medium containing DMEM HighGlucose (Gibco), 10% fetal bovine serum (FBS from Gibco), 1×non-essential amino acid (NEAA from Gibco) and 1×Penicillin/Streptomycine/Glutamine (Pen/Strep/Glu from Gibco). 5.6 μgeach of Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], andFc-L10-OSK1[K7S,E16K,K20D] in pCMVi plasmid that had beenphenol/chloroform extracted was transfected into HEK-293 cells usingFuGENE 6 (Roche). The cells were recovered for 24 hours, and then placedin DMEM High Glucose, 1x NEAA and 1× Pen/Strep/Glu medium for 48 hours.Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D]were purified from medium conditioned by these transfected HEK-293 cellsusing a protocol described in Example 50 herein below.

Fifteen μl of conditioned medium was mixed with an in-house 4× LoadingBuffer (without β-mercaptoethanol) and electrophoresed on a Novex 4-20%tris-glycine gel using a Novex Xcell II apparatus at 101V/46 mA for 2hours in a 1× Gel Running solution (25 mM Tris Base, 192 mM Glycine, 3.5mM SDS) along with 20 μl of BenchMark Pre-Stained Protein ladder(Invitrogen). The gel was then soaked in Electroblot buffer (25 mM Trisbase, 192 mM glycine, 20% methanol,) for 5 minutes. -A nitrocellulosemembrane from Invitrogen (Cat. No. LC200, 0.2 μm pores size) was soakedin Electroblot buffer. The pre-soaked gel was blotted to thenitrocellulose membrane using the Mini Trans-Blot Cell module accordingto the manufacturer instructions (Bio-Rad Laboratories) at 300 mA for 2hours. The blot was rinsed in Tris buffered saline solution pH7.5 with0.1% Tween20 (TBST). Then, the blot was first soaked in a 5% milk(Camation) in TBST for 1 hour at room temperature, followed by washingthree times in TBST for 10 minutes per wash. Then, incubated with 1:1000dilution of the HRP-conjugated Goat anti-human IgG, (Fcγ) antibody(Pierece Biotechnology Cat. no. 31413) in TBST with 5% milk buffer for 1hour with shaking at room temperature. The blot was then washed threetimes in TBST for 15 minutes per wash at room temperature. The primaryantibody was detected using Amersham Pharmacia Biotech's ECL westernblotting detection reagents according to manufacturers instructions.Upon ECL detection, the western blot analysis displayed the expectedsize of 66 kDa under non-reducing gel conditions (FIG. 46).

Plasmids containing the Fc-L10-OSK1, Fc-L10-OSK1[K7S],Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] inserts in pCMVivector were digested with XbaI and NotI (Roche) restriction enzymes andgel purified. The inserts were individually ligated into SpeI and NotI(Roche) digested pDSRα24 (Amgen Proprietary) expression vector.Integrity of the resulting constructs were confirmed by DNA sequencing.Although, analysis of several sequences of clones yielded a 100% percentmatch with the above sequence, only one clone was selected for largescaled plasmid purification.

AM1 CHOd- (Amgen Proprietary) cells used in the stable expression ofFc-L10-OSK1 protein were cultured in AM1 CHOd- growth medium containingDMEM High Glucose, 10% fetal bovine serum, 1× hypoxantine/thymidine (HTfrom Gibco), 1×NEAA and 1× Pen/Strep/Glu. 5.6 μg of pDSRα-24-Fc-L10-OSK1plasmid was transfected into AM1 CHOd- cells using FuGene 6. Twenty-fourhours post transfection, the cells were split 1:11 into DHFR selectionmedium (DMEM High Glucose plus 10% Dialyzed Fetal Bovine Serum (dFBS),1×NEAA and 1× Pen/Strep/Glu) at 1:50 dilution for colony selection. Thecells were selected in DHFR selection medium for thirteen days. The ten10-cm² pools of the resulting colonies were expanded to ten T-175flasks, then were scaled up ten roller bottles and cultured under AM1CHOd- production medium (DMEM/F12 (1:1), 1×NEAA, 1× Sodium Pyruvate (NaPyruvate), 1× Pen/Strep/Glu and 1.5% DMSO). The conditioned medium washarvested and replaced at one-week intervals. The resulting six litersof conditioned medium were filtered through a 0.45 μm cellulose acetatefilter (Corning, Acton, Mass.), and characterized by SDS-PAGE analysisas shown in FIG. 47. Then, transferred to Protein Chemistry forpurification.

Twelve colonies were selected after 13 days on DHFR selection medium andpicked into one 24-well plate. The plate was allowed to grow up for oneweek, and then was transferred to AM1 CHOd- production medium for 48-72hours and the conditioned medium was harvested. The expression levelswere evaluated by Western blotting similar to the transient Western blotanalysis with detection by the same HRP-conjugated Goat anti-human IgG,(Fcγ) antibody to screen 5 μl of conditioned medium. All 12 stableclones exhibited expression at the expected size of 66 kDa. Two clones,A3 and C2 were selected and expanded to T175 flask for freezing with A3as a backup to the primary clone C2 (FIG. 48).

The C2 clone was scaled up into fifty roller bottles (Corning) usingselection medium and grown to confluency. Then, the medium was exchangedwith a production medium, and let incubate for one week. The conditionedmedium was harvested and replaced at the one-week interval. Theresulting fifty liters of conditioned medium were filtered through a0.45 μm cellulose acetate filter (Corning, Acton, Mass.), andcharacterized by SDS-PAGE analysis (data not shown). Furtherpurification was accomplished as described in Example 42 herein below.

Example 42 Purification of Fc-L10-OSK1, Fc-L10-OSK1(K7S),Fc-L10-OSK1(E16K,K20D), and Fc-L10-OSK1(K7S,E16K,K20D) Expressed byMammalian Cells

Purification of Fc-L10-OSK1. Approximately 6 L of CHO (AM1 CHOd-)cell-conditioned medium (see, Example 41 above) was loaded on to a 35 mlMAb Select column (GE Healthcare) at 10 ml/min 7° C., and the column waswashed with several column volumes of Dulbecco's phosphate bufferedsaline without divalent cations (PBS) and sample was eluted with a stepto 100 mM glycine pH 3.0. The MAb Select elution was directly loaded onto an inline 65 ml SP-HP column (GE Healthcare) in S-Buffer A (20 mMNaH₂PO₄, pH 7.0) at 10 ml/min 7° C. After disconnecting the MAb selectcolumn, the SP-HP column was then washed with several column volumesS-Buffer A, and then developed using a linear gradient from 5% to 60%S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.0) at 10 ml/min followed by astep to 100% S-Buffer B at 7° C. Fractions were then analyzed using aCoomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and thefractions containing the desired product were pooled based on thesedata. The pooled material was then concentrated to about 20 ml using aPall Life Sciences Jumbosep 10K Omega centrifugal ultra-filtrationdevice. The concentrated material was then buffer exchanged by dilutingwith 20 ml of 20 mM NaH₂PO₄, pH 7.0 and reconcentrated to 20 ml usingthe Jumbosep 10K Omega filter. The material was then diluted with 20 ml20 mM NaH₂PO₄, 200 mM NaCl, pH 7.0 and then reconcentrated to 22 ml. Thebuffer exchanged material was then filtered though a Pall Life SciencesAcrodisc with a 0.22 μm, 25 mm Mustang E membrane at 1 ml/min roomtemperature. A spectral scan was then conducted on 50 μl of the filteredmaterial diluted in 700 μl PBS using a Hewlett Packard 8453spectrophotometer (FIG. 49A, black trace). The concentration of thefiltered material was determined to be 4.96 mg/ml using a calculatedmolecular mass of 30,371 g/mol and extinction coefficient of 35,410 M⁻¹cm⁻¹. The purity of the filtered material was then assessed using aCoomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 49B).The endotoxin level was then determined using a Charles RiverLaboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a30-fold dilution of the sample in Charles Rivers Laboratories EndotoxinSpecific Buffer yielding a result of 1.8 EU/mg protein. Themacromolecular state of the product was then determined using sizeexclusion chromatography on 149 μg of the product injected on to aPhenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mMNaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 49C).The product was then subject to mass spectral analysis by diluting 1 μlof the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05%trifluoroacetic acid, 50% acetonitrile). One milliliter of the resultantsolution was spotted onto a MALDI sample plate. The sample was allowedto dry before being analyzed using a Voyager DE-RP time-of-flight massspectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). Thepositive ion/linear mode was used, with an accelerating voltage of 25kV. Each spectrum was produced by accumulating data from about 200 lasershots. External mass calibration was accomplished using purifiedproteins of known molecular masses. (FIG. 49D). The product was thenstored at −80° C.

The yield for the mammalian Fc-L10-OSK1 prep was 115 mg from 6 L, thepurity was >90% judging by SDS-PAGE; Fc-L10-OSK1 ran as the expecteddimer judging by SEC-HPLC, and the mass is with the expected rangejudging by MS.

The activity of purified Fc-L10-OSK1 in blocking human Kv1.3 and humanKv1.1 is described in Example 43 herein below.

Purification of Fc-L10-OSK1(K7S). Fc-L10-OSK1(E16K,K20D), andFc-L10-OSK1(K7S,E16K,K20D). Approximately 500 mL of medium conditionedby transfected HEK-293 (see, Example 41 above) was combined with a 65%slurry of MAb Select resin (1.5 ml) (GE Healthcare) and 500 μl 20% NaN₃.The slurry was then gently agitated for 3 days at 4° C. followed bycentrifugation at 1000 g for 5 minutes at 4° C. using no brake. Themajority of the supernatant was then aspirated and the remaining slurryin the pellet was transferred to a 14 ml conical tube and combined with12 ml of Dulbecco's phosphate buffered saline without divalent cations(PBS). The slurry was centrifuged at 2000 g for 1 minute at 4° C. usinga low brake and the supernatant was aspirated. The PBS wash cycle wasrepeated an additional 3 times. The bound protein was then eluted byadding 1 ml of 100 mM glycine pH 3.0 and gently agitating for 5 min atroom temperature. The slurry was then centrifuged at 2000 g for 1 minuteat 4° C. using a low brake and the supernatant was aspirated as thefirst elution. The elution cycle was repeated 2 more times, and all 3supernatants were combined into a single pool. Sodium acetate (37.5 μlof a 3 M solution) was added to the elution pool to raise the pH, whichwas then dialyzed against 10 mM acetic acid, 5% sorbitol, pH 5.0 for 2hours at room temperature using a 10 kDa SlideAlyzer (Pierce). Thedialysis buffer was changed, and the dialysis continued over night at 4°C. The dialyzed material was then filtered through a 0.22 μm celluloseacetate filter syringe filter. Then concentration of the filteredmaterial was determined to be 1.27 mg/ml using a calculated molecularmass of 30,330 and extinction coefficient of 35,410 M⁻¹ cm⁻¹ (FIG. 50A).The purity of the filtered material was then assessed using a Coomassiebrilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 50B). Theendotoxin level was then determined using a Charles River LaboratoriesEndosafe-PTS system (0.05-5 EU/ml sensitivity) using a 25-fold dilutionof the sample in Charles Rivers Laboratories Endotoxin Specific Bufferyielding a result of <1 EU/mg protein. The macromolecular state of theproduct was then determined using size exclusion chromatography on 50 μgof the product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm (FIG. 50C). The product was then subject tomass spectral analysis by diluting 1 μl of the sample into 10 μl ofsinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50%acetonitrile). One milliter of the resultant solution was spotted onto aMALDI sample plate. The sample was allowed to dry before being analyzedusing a Voyager DE-RP time-of-flight mass spectrometer equipped with anitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode wasused, with an accelerating voltage of 25 kV. Each spectrum was producedby accumulating data from ˜200 laser shots. External mass calibrationwas accomplished using purified proteins of known molecular masses.(FIG. 50D). The product was then stored at −80° C.

FIGS. 51A-D show results from the purification and analysis forFc-L10-OsK1(E16K, K20D), which was conducted using the same protocol asthat for the Fc-L110-OsK1 (K7S) molecule (described above) with thefollowing exceptions: the concentration was found to be 1.59 mg/ml usinga calculated molecular mass of 30,357 g/mol and a calculated extinctioncoefficient of 35,410; the pyrogen level was found to be <1 EU/mg usinga 32-fold dilution.

FIGS. 52A-D show results from the purification and analysis forFc-L10-OsK1(K7S,E16K, K20D), which was conducted using the same protocolas that for the Fc-L10-OsK1(K7S) molecule (described above) with thefollowing exceptions: the concentration was found to be 0.81 mg/ml usinga calculated molecular mass of 30,316 g/mol and a calculated extinctioncoefficient of 35,410; the pyrogen level was found to be <1 EU/mg usinga 16-fold dilution.

The activity of purified Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K, K20D] andFc-L10-OSK1[K7S, E16K, K20D] in blocking human Kv1.3 and human Kv1.1 isdescribed in Example 43 herein below.

Example 43 Electrophysiology of OSK1 and OSK1 Peptibody Analogs

A 38-residue peptide toxin of the Asian scorpion Orthochirusscrobiculosus venom (OSK1) was synthesized (see, Examples 41) toevaluate its impact on the human Kv1.1 and Kv1.3 channels, subtypes ofthe potassium channel family. The potency and selectivity of syntheticOSK1 in inhibiting the human Kv1.1 and Kv1.3 channels was evaluated bythe use of HEK293 cell expression system and electrophysiology (FIG.53). Whole cell patch clamp recording of stably expressed Kv1.3 channelsrevealed that the synthetic OSK1 peptide is more potent in inhibitinghuman Kv1.3 when compared to Kv1.1 (Table 33).

Fusion of OSK1 peptide toxin to antibody to generate OSK1 peptibody. Toimprove plasma half-life and prevent OSK1 peptide toxin from penetratingthe CNS, the OSK1 peptide toxin was fused to the Fc-fragment of a humanantibody IgG1 via a linker chain length of 10 amino acid residues(Fc-L10-OSK1), as described in Example 41 herein. This fusion resultedin a decrease in the potency of Kv1.3 by 5-fold when compared to thesynthetic OSK1 peptide. However, it significantly improved theselectivity of OSK1 against Kv1.1 by 210-fold when compared to that ofthe synthetic peptide alone (4-fold; Table 33 and FIG. 54).

Modification of OSK1-peptibody (Fc-L10-OSK1). OSK1 shares 60 to 80%sequence homology to other members of scorpion toxins, which arecollectively termed α-KTx3. Sequence alignment of OSK1 and other membersof α-KTx3 family revealed 4 distinct structural differences at positions12, 16, 20, and 36. These structural differences of OSK1 have beenpostulated to play an important role in its wide range of activitiesagainst other potassium channels, which is not observed with othermembers of α-KTx3 family. Hence, two amino acid residues at position 16and 20 were restored to the more conserved amino acid residues withinthe OSK1 sequence in order to evaluate their impact on selectivityagainst other potassium channels such as Kv1.1, which is predominantlyfound in the CNS as a heterotetromer with Kv1.2. By substituting forglutamic acid at position 16, and for lysine at position 20, theconserved lysine and aspartic acid residues, respectively (i.e.,Fc-L10-OSK1[E16K, K20D]), we did not observe a significant change inpotency when compared to that of Fc-L10-OSK1 (1.3-fold difference; FIG.56 and Table 33). However, this double mutation removed the blockingactivity against Kv1.1. The selectivity ratio of Kv1.1/Kv1.3 was403-fold, which was a significant improvement over the selectivity ratiofor Fc-L10-OSK1 (210-fold). A single amino acid mutation at position 7from lysine to serine (Fc-L10-OSK1[K7S]) produced a slight change inpotency and selectivity by 2- and 1.3-fold, respectively, when comparedto those of Fc-L10-OSK1 (FIG. 55 and Table 33). There was a significantdecrease in potency as well as selectivity when all three residues weremutated to generate Fc-L10-OSK1[K7S, E16K, K20D] (FIG. 57 and Table 33).

As demonstrated by the results in Table 33, we dramatically improvedselectivity against Kv1.1 by fusing the OSK1 peptide toxin to theFc-fragment of the human antibody IgG1, but reduced target potencyagainst Kv1.3. The selectivity against Kv1.1 was further improved when 2residues at two key positions were restored to the conserved residuesfound in other members of the α-KTx3 family.

Table 33 shows a summary of IC50 values for OSK1 and OSK1 analogsagainst hKv1.3 and hKv1.1 channels. All analogues are ranked based ontheir potency against hKv1.3. Also shown in the table is the selectivityratio of hKv1.1/hKv1.3 for all OSK1 analogs.

hKv1.3: hKv1.1: IC₅₀ IC₅₀ hKv1.1/ Compound [pM] [pM] hKv1.3 SyntheticOSK1 39 160 4 Fc-L10-OSK1 198 41600 210 Fc-L10-OSK1[E16K, K20D] 248100000 403 Fc-L10-OSK1[K7S] 372 100000 269 Fc-L10-OSK1[K7S, E16K, K20D]812 10000 12

Example 44 Pharmacokinetic Study of PEG-ShK[1-35] Molecule in Rats

The intravenous (IV) pharmacokinetic profile was determined of a about24-kDa 20K PEG-ShK[1-35] molecule and the about 4-kDa small native ShKpeptide was determined in Spraque Dawley rats. The IV dose for thenative ShK peptide and our novel 20K PEG-ShK[1-35] molecule was 1 mg/kg.This dose represented equal molar amounts of these two molecules. Theaverage weight of the rats was about 0.3 kg and two rats were used foreach dose & molecule. At various times following IV injection, blood wasdrawn and about 0.1 ml of serum was collected. Serum samples were storedfrozen at −80° C. until analysis.

Assay Plate preparation for electrophysiology. Rat serum samplescontaining the 20K PEG-ShK[1-35] molecule or the native ShK peptide frompharmacokinetic studies were received frozen. Before experiments, eachsample was thawed at room temperature and an aliquot (70 to 80 μl) wastransferred to a well in a 96-well polypropylene plate. In order toprepare the Assay Plate, several dilutions were made from thepharmacokinetic serum samples to give rise to Test Solutions. Dilutionsof serum samples from the pharmacokinetic study were into 10% PhosphateBuffered Saline (PBS, with Ca², and Mg²⁺). For determination of theamount of our novel 20K PEG-ShK[1-35] molecule in serum samples from thepharmacokinetic study, the final serum concentrations in the TestSolutions were 90%, 30%, 10%, 3.3% and 1.1%. Purified 20K PEG-Shk[1-35]Standard inhibition curves were also prepared in the Assay Plate. To dothis, 8-point serial dilutions of the purified 20K PEG-ShK[1-35]molecule (Standard) were prepared in either 90%, 30%, 10%, 3.3% or 1.1%rat serum and the final concentration of standard was 50, 16.7, 5.5,1.85, 0.62, 0.21, 0.068 and 0.023 nM.

Cell preparation for electrophysiology. CHO cells stably expressing thevoltage-activated K⁺ channel, K_(V)1.3 were plated in T-175 tissueculture flasks (at a density of 5×10⁶) 2 days before experimentation andallowed to grow to around 95% confluence. Immediately prior to theexperiment, the cells were washed with PBS and then detached with a 2 mlmixture (1:1 volume ratio) of trypsin (0.25%) and versene (1:5000) at37° C. (for 3 minutes). Subsequently, the cells were re-suspended in theflask in 10 ml of tissue culture medium (HAM's F-12 with Glutamax,Invitrogen, Cat#31765) with 10% FBS, 1×NEAA and 750 μg/ml of G418) andcentrifuged at about 1000 rpm for 1½ minutes. The resultant cell pelletwas re-suspended in PBS at 3-5×10⁶ cells/ml.

IonWorks electrophysiology and data analysis. The ability of Testsolutions or Standards in serum to inhibit K⁺ currents in the CHO-Kv1.3cells was investigated using the automated electrophysiology system,IonWorks Quattro. Re-suspended cells, the Assay Plate, a PopulationPatch Clamp (PPC) PatchPlate as well as appropriate intracellular (90mMK-Gluconate, 20 mMKF, 2 mM NaCl, 1 mM MgCl2, 10 mM EGTA, 10 mM HEPES,pH 7.35) and extracellular (PBS, with Ca²⁺ and Mg²⁺) buffers werepositioned on IonWorks Quattro. Electrophysiology recordings were madefrom the CHO-Kv1.3 cells using an amphotericin-based perforatedpatch-clamp method. Using the voltage-clamp circuitry of the IonWorksQuattro, cells were held at a membrane potential of −80 mV andvoltage-activated K⁺ currents were evoked by stepping the membranepotential to +30 mV for 400 ms. K⁺ currents were evoked under controlconditions i.e., in the absence of inhibitor at the beginning of theexperiment and after 10-minute incubation in the presence of the TestSolution or Standard. The mean K⁺ current amplitude was measured between430 and 440 ms and the data were exported to a Microsoft Excelspreadsheet. The amplitude of the K⁺ current in the presence of eachconcentration of the Test Solution or Standard was expressed as apercentage of the K⁺ current in control conditions in the same well.

Standard inhibition curves were generated for each standard in variouslevels of rat serum and expressed as current percent of control (POC)versus log of nM concentration. Percent of control (POC) is inverselyrelated to inhibition, where 100 POC is no inhibition and 0 POC is 100%inhibition. Linear regression over a selected region of the curve wasused to derive an equation to enable calculation of drug concentrationswithin Test solutions. Only current values within the linear portion ofthe Standard curve were used to calculate the concentration of drug inTest solutions. The corresponding Standard curve in a given level ofserum, was always compared to the same level of serum of Test solutionwhen calculating drug level. The Standard curves for ShK and 20KPEG-ShK[1-35] are shown in FIG. 58A and FIG. 58B, respectively, and eachfigure contains linear regression equations for each Standard at a givenpercentage of serum. For the 20K PEG-ShK[1-35] standard curve the linearportion of the Standard curve was from 20 POC to 70 POC and only currentvalues derived from the Test solution which fell within this range wereused to calculate drug concentration within the Test solution.

The pharmacokinetic profile of our novel 20K PEG ShK[1-35] moleculeafter IV injection is shown in FIG. 59. The terminal half-life (t_(1/2)b) of this molecule is estimated from this curve to be between 6 to 12hours long. Beyond 48 hours, the level of drug falls outside the linearrange of the Standard curve and is not calculated. The calculated 6 to12 hour half-life of our novel 20K PEG-ShK[1-35] molecule wassubstantially longer than the approximately 0.33 hour (or 20 min)half-life of the native ShK molecule reported earlier by C. Beeton etal. [C. Beeton et al. (2001) Proc. Natl. Acad. Sci. 98, 13942-13947],and is a desirable feature of a therapeutic molecule. A comparison ofthe relative levels of Kv1.3 inhibitor after an equal molar IV injectionof ShK versus 20K PEG-ShK[1-35] is shown in FIG. 60. As can be seen fromthis figure examining 5% serum Test solutions, the 20K PEG-ShK[1-35]molecule showed significant suppression of Kv1.3 current (<70 POC) formore than 24 hours, whereas the native ShK peptide only showed asignificant level of inhibition of Kv1.3 current for the first hour andbeyond 1 hour showed no significant blockade. These data againdemonstrate a desirable feature of the 20K PEG ShK[1-35] molecule as atherapeutic for treatment of autoimmune disease.

Example 45 PEGylated Toxin Peptide Suppressed Severe AutoimmuneEncephalomyelitis in Animal Model

The 20KPEG-ShK inhibitor of Kv1.3 shows improved efficacy in suppressingsevere autoimmune encephalomyelitis in rats. Using an adoptive transferexperimental autoimmune encephalomyelitis (AT-EAE) model of multiplesclerosis described earlier [C. Beeton et al. (2001) J. Immunol. 166,936], we examined the activity in vivo of our novel 20KPEG-ShK moleculeand compared its efficacy to that of the ShK toxin peptide alone. Thestudy design is illustrated in FIG. 61. The results from this in vivostudy are provided in FIG. 62 and FIG. 63. The 20KPEG-ShK moleculedelivered subcutaneously (SC) at 10 μg/kg daily from day −1 to day 3significantly reduced disease severity and increased survival, whereasanimals treated with an equal molar dose (10 μg/kg) of the small ShKpeptide developed severe disease and died.

The 35-amino acid toxin peptide ShK (Stichodactyla helianthusneurotoxin) was purchased from Bachem Bioscience Inc and confirmed byelectrophysiology to potently block Kv1.3 (see Example 36 herein). Thesynthesis, PEGylation and purification of the 20KPEG ShK molecule was asdescribed herein above. The encephalomyelogenic CD4+ rat T cell line,PAS, specific for myelin-basic protein (MBP) originated from Dr. EvelyneBeraud. The maintenance of these cells in vitro and their use in theAT-EAE model has been described earlier [C. Beeton et al. (2001) PNAS98, 13942]. PAS T cells were maintained in vitro by alternating roundsof antigen stimulation or activation with MBP and irradiated thymocytes(2 days), and propagation with T cell growth factors (5 days).Activation of PAS T cells (3×10⁵/ml) involved incubating the cells for 2days with 10 μg/ml MBP and 15×10⁶/ml syngeneic irradiated (3500 rad)thymocytes. On day 2 after in vitro activation, 10-15×10⁶ viable PAS Tcells were injected into 6-12 week old female Lewis rats (Charles RiverLaboratories) by tail IV. Daily subcutaneous injections of vehicle (2%Lewis rat serum in PBS), 20KPEG-ShK or ShK were given from days −1 to 3(FIG. 61), where day −1 represent 1 day prior to injection of PAS Tcells (day 0). In vehicle treated rats, acute EAE developed 4 to 5 daysafter injection of PAS T cells (FIG. 62). Serum was collected byretro-orbital bleeding at day 4 and by cardiac puncture at day 8 (end ofthe study) for analysis of levels of inhibitor. Rats were weighed ondays −1, 4, 6, and 8. Animals were scored blinded once a day from theday of cell transfer (day 0) to day 3, and twice a day from day 4 to day8. Clinical signs were evaluated as the total score of the degree ofparesis of each limb and tail. Clinical scoring: 0=No signs, 0.5=distallimp tail, 1.0=limp tail, 2.0=mild paraparesis, ataxia, 3.0=moderateparaparesis, 3.5=one hind leg paralysis, 4.0=complete hind legparalysis, 5.0=complete hind leg paralysis and incontinence,5.5=tetraplegia, 6.0=moribund state or death. Rats reaching a score of5.5 were euthanized.

Treatment of rats with the Kv1.3 blocker PEG-ShK prior to the onset ofEAE caused a lag in the onset of disease, inhibited the progression ofdisease, and prevented death in a dose-dependent manner (FIG. 62). Onsetof disease in rats that were treated with the vehicle alone, 10 μg/kgShK or 1 μg/kg of PEG-ShK was observed on day 4, compared to day 4.5 inrats treated with 10 μg/kg PEG-ShK or 100 μg/kg PEG-ShK. In addition,rats treated with vehicle alone, 10 μg/kg ShK or 1 μg/kg of PEG-ShK alldeveloped severe disease by the end of the study with an EAE score of5.5 or above. In contrast, rats treated with 10 μg/kg PEG-ShK or 100μg/kg PEG-ShK, reached a peak clinical severity score average of <2, andall but one rat survived to the end of the study. Furthermore, we foundthat rat body weight correlated with disease severity (FIG. 63). Ratstreated with vehicle alone, 10 μg/kg ShK or 1 μg/kg of PEG-ShK all lostan average of 31 g, 30 g, and 30 g, respectively, while rats treatedwith 10 μg/kg PEG-ShK or 100 μg/kg PEG-ShK lost 18 g and 11 g,respectively. Rats in the latter two groups also appeared to be gainingweight by the end of the study, a sign of recovery. It should be notedthat rats treated with 10 μg/kg ShK and 10 μg/kg PEG-ShK received molarequivalents of the ShK peptide. The significantly greater efficacy ofthe PEG-ShK molecule relative to unconjugated ShK, is likely due to thePEG-ShK molecule's greater stability and prolonged half-life in vivo(see, Example 44).

Example 46 Compositions Including Kv1.3 Antagonist Peptides BlockInflammation in Human Whole Blood

Ex vivo assay to examine impact of Kv1.3 inhibitors on secretion of IL-2and IFN-g. Human whole blood was obtained from healthy, non-medicateddonors in a heparin vacutainer. DMEM complete media was Iscoves DMEM(with L-glutamine and 25 mM Hepes buffer) containg 0.1% human albumin(Bayer #68471), 55 μM 2-mercaptoethanol (Gibco), and 1× Pen-Strep-Gln(PSG, Gibco, Cat#10378-016). Thapsigargin was obtained from Alomone Labs(Israel). A 10 mM stock solution of thapsigargin in 100% DMSO wasdiluted with DMEM complete media to a 40 μM, 4× solution to provide the4× thapsigargin stimulus for calcium mobilization. The Kv1.3 inhibitorpeptide ShK (Stichodacytla helianthus toxin, Cat# H2358) and the BKCalinhibitor peptide IbTx (Iberiotoxin, Cat# H9940) were purchased fromBachem Biosciences, whereas the Kv1.1 inhibitor peptide DTX-k(Dendrotoxin-K) was from Alomone Labs (Israel). The CHO-derivedFc-L10-ShK[2-35] peptibody inhibitor of Kv1.3 was obtained as describedherein at Example 4 and Example 39. The calcineurin inhibitorcyclosporin A was obtained from the Amgen sample bank, but is alsoavailable commercially from a variety of vendors. Ten 3-fold serialdilutions of inhibitors were prepared in DMEM complete media at 4× finalconcentration and 50 μl of each were added to wells of a 96-well Falcon3075 flat-bottom microtiter plate. Whereas columns 1-5 and 7-11 of themicrotiter plate contained inhibitors (each row with a separateinhibitor dilution series), 50 μof DMEM complete media alone was addedto the 8 wells in column 6 and 100 μof DMEM complete media alone wasadded to the 8 wells in column 12. To initiate the experiment, 100 μofwhole blood was added to each well of the microtiter plate. The platewas then incubated at 37° C, 5% CO₂ for one hour. After one hour, theplate was removed and 50 μof the 4× thapsigargin stimulus (40 μM) wasadded to all wells of the plate, except the 8 wells in column 12. Theplates were placed back at 37° C, 5% CO₂ for 48 hours. To determine theamount of IL-2 and IFN-g secreted in whole blood, 100 μl of thesupernatant (conditioned media) from each well of the 96-well plate wastransferred to a storage plate. For MSD electrochemilluminesenceanalysis of cytokine production, 20 μl of the supernatants (conditionedmedia) were added to MSD Multi-Spot Custom Coated plates(meso-scale.com). The working electrodes on these plates were coatedwith four Capture Antibodies (hIL-5, hIL-2, hIFNg and hIL-4) in advance.After addition of 20 μl of conditioned media to the MSD plate, 150 μof acocktail of Detection Antibodies and P4 Buffer were added to each well.The 150 μcocktail contained 20 μl of four Detection Antibodies (hIL-5,hIFNg and hIL-4) at 1 μg/ml each and 130 μl of 2× P4 Buffer. The plateswere covered and placed on a shaking platform overnight (in the dark).The next morning the plates were read on the MSD Sector Imager. Sincethe 8 wells in column 6 of each plate received only the thapsigarginstimulus and no inhibitor, the average MSD response here was used tocalculate the “High” value for a plate. The calculate “Low” value forthe plate was derived from the average MSD response from the 8 wells incolumn 12 which contained no thapsigargin stimulus and no inhibitor.Percent of control (POC) is a measure of the response relative to theunstimulated versus stimulated controls, where 100 POC is equivalent tothe average response of thapsigargin stimulus alone or the “High” value.Therefore, 100 POC represents 0% inhibition of the response. Incontrast, 0 POC represents 100% inhibition of the response and would beequivalent to the response where no stimulus is given or the “Low”value. To calculate percent of control (POC), the following formula isused: [(MSD response of well)−(“Low”)]/[(“High”)−(“Low”)]×100. Thepotency of the molecules in whole blood was calculated after curvefitting from the inhibition curve (IC) and IC50 was derived usingstandard curve fitting software. Although we describe here measurementof cytokine production using a high throughput MSDelectrochemillumenescence assay, one of skill in the art can readilyenvision lower throughput ELISA assays are equally applicable formeasuring cytokine production.

Ex vivo assay demonstrating Kv1.3 inhibitors block cell surfaceactivation of CD40L & IL-2R. Human whole blood was obtained fromhealthy, non-medicated donors in a heparin vacutainer. DMEM completemedia was Iscoves DMEM (with L-glutamine and 25 mM Hepes buffer)containing 0.1% human albumin (Bayer #68471), 55 μM 2-mercaptoethanol(Gibco), and 1× Pen-Strep-Gln (PSG, Gibco, Cat#10378-016). Thapsigarginwas obtained from Alomone Labs (Israel). A 10 mM stock solution ofthapsigargin in 100% DMSO was diluted with DMEM complete media to a 40μM, 4× solution to provide the 4× thapsigargin stimulus for calciummobilization. The Kv1.3 inhibitor peptide ShK (Stichodacytla helianthustoxin, Cat# H2358) and the BKCa1 inhibitor peptide IbTx (Iberiotoxin,Cat# H9940) were purchased from Bachem Biosciences, whereas the Kv1.1inhibitor peptide DTX-k (Dendrotoxin-K) was from Alomone Labs (Israel).The CHO-derived Fc-L110-ShK[2-35] peptibody inhibitor of Kv1.3 wasobtained as described in Example 4 and Example 39. The calcineurininhibitor cyclosporin A was obtained from the Amgen sample bank, but isalso available commercially from a variety of vendors. The ion channelinhibitors ShK, IbTx or DTK-k were diluted into DMEM complete media to4× of the final concentration desired (final=50 or 100 nM). Thecalcineurin inhibitor cyclosporin A was also diluted into DMEM completemedia to 4× final concentration (final=10 μM). To appropriate wells of a96-well Falcon 3075 flat-bottom microtiter plate, 50 μl of either DMEMcomplete media or the 4× inhibitor solutions were added. Then, 100 μl ofhuman whole blood was added and the plate was incubated for 1 hour at37° C., 5% CO₂. After one hour, the plate was removed and 50 μl of the4× thapsigargin stimulus (40 μM) was added to all wells of the platecontaining inhibitor. To some wells containing no inhibitor but justDMEM complete media, thapsigargin was also added whereas others wellswith just DMEM complete media had an additional 50 μl of DMEM completemedia added. The wells with no inhibitor and no thapsigargin stimulusrepresented the untreated “Low” control. The wells with no inhibitor butwhich received thapsigargin stimulus represented the control for maximumstimulation or “High” control. Plates were placed back at 37° C., 5% CO₂for 24 hours. After 24 hours, plates were removed and wells were processfor FACS analysis. Cells were removed from the wells and washed instaining buffer (phosphate buffered saline containing 2%heat-inactivated fetal calf serum). Red blood cells were lysed using BDFACS Lysing Solution containing 1.5% formaldehyde (BD Biosciences) asdirected by the manufacturer. Cells were distributed at a concentrationof 1 million cells per 100 microliters of staining buffer per tube.Cells were first stained with 1 microliter of biotin-labeled anti-humanCD4, washed, then stained simultaneously 1 microliter each ofstreptavidin-APC, FITC-labeled anti-human CD45RA, and phycoerythrin(PE)-labeled anti-human CD25 (IL-2Ra) or PE-labeled anti-human CD40L.Cells were washed with staining buffer between antibody addition steps.All antibodies were obtained from BD Biosciences (San Diego, Calif.).Twenty to fifty thousand live events were collected for each sample on aBecton Dickinson FACSCaliber (Mountain View, Calif.) flow cytometer andanalyzed using FlowJo software (Tree Star Inc., San Carlos, Calif.).Dead cells, monocytes, and granulocytes were excluded from the analysison the basis of forward and side scatter properties.

FIG. 64 and FIG. 67 demonstrate that Kv1.3 inhibitors ShK andFc-L10-ShK[2-35] potently blocked IL-2 secretion in human whole blood,in addition to suppressing activation of the IL-2R on CD4+ T cells. TheKv1.3 inhibitor Fc-L10-ShK[2-35] was more than 200 times more potent inblocking IL-2 production in human whole blood than cyclosporine A (FIG.64) as reflected by the IC50. FIG. 65 shows that Kv1.3 inhibitors alsopotently blocked secretion of IFNg in human whole blood, and FIG. 66demonstrates that upregulation of CD40L on T cells was additionallyblocked. The data in FIGS. 64-67 show that the Fc-L10-ShK[2-35] moleculewas stable in whole blood at 37° C. for up to 48 hours, providing potentblockade of inflammatory responses. Toxin peptide therapeutic agentsthat target Kv1.3 and have prolonged half-life, are sought to providesustained blockade of these responses in vivo over time. In contrast,despite the fact the Kv1.3 inhibitor peptide ShK also showed potentblockade in whole blood, the ShK peptide has a short (˜20 min) half-lifein vivo (C. Beeton et al. (2001) Proc. Natl. Acad. Sci. 98, 13942), andcannot, therefore, provide prolonged blockade. Whole blood represents aphysiologically relevant assay to predict the response in animals. Thewhole blood assays described here can also be used as a pharmacodynamic(PD) assay to measure target coverage and drug exposure following dosingof patients. These human whole blood data support the therapeuticusefulness of the compositions of the present invention for treatment ofa variety immune disorders, such as multiple sclerosis, type 1 diabetes,psoriasis, inflammatory bowel disease, contact-mediated dermatitis,rheumatoid arthritis, psoriatic arthritis, asthma, allergy, restinosis,systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogrensyndrome, inflammatory bone resorption, transplant rejection,graft-versus-host disease, and lupus.

Example 47 PEGylated Peptibodies

By way of example, PEGylated peptibodies of the present invention weremade by the following method. CHO-expressed FcL10-OsK1 (19.2 mg; MW30,371 Da, 0.63 micromole) in 19.2 ml A5S, 20 mM NaBH₃CN, pH 5, wastreated with 38 mg PEG aldehyde (MW 20 kDa; 3×, Lot 104086). The sealedreaction mixture was stirred in a cold room overnight. The extent of theprotein modification during the course of the reaction was monitored bySEC HPLC using a Superose 6 HR 10/30 column (Amersham Pharmacia Biotech)eluted with a 0.05 M phosphate buffer, 0.5 M NaCl, pH 7.0 at 0.4 ml/min.The reaction mixture was dialyzed with A5S, pH 5 overnight. The dialyzedmaterial was then loaded onto an SP HP FPLC column (16/10) in A5S pH 5and eluted with a 1 M NaCl gradient. The collected fractions wereanalyzed by SEC HPLC, pooled into 3 pools, exchanged into DPBS,concentrated and submitted for functional testing (Table 34).

In another example, FcL10-ShK1 (16.5 mg; MW 30,065 Da, 0.55 micro mole)in 16.5 ml A5S, 20 mM NaBH₃CN, pH 5 was treated with 44 mg PEG aldehyde(MW 20 kDa; 4×, Lot 104086). The sealed reaction mixture was stirred ina cold room overnight. The extent of the protein modification during thecourse of the reaction was monitored by SEC HPLC using a Superose 6 HR10/30 column (Amersham Pharmacia Biotech) eluted with a 0.05 M phosphatebuffer, 0.5 M NaCl, pH 7.0 at 0.4 ml/min. The reaction mixture wasdialyzed with A5S, pH 5 overnight. The dialyzed material was loaded ontoan SP HP FPLC column (16/10) in A5S pH 5 and was eluted with a 1 M NaClgradient. The collected fractions were analyzed by SEC HPLC, pooled into3 pools, exchanged into DPBS, concentrated and submitted for functionaltesting (Table 34).

The data in Table 34 demonstrate potency of the PEGylated peptibodymolecules as Kv1.3 inhibitors.

Table 34 shows determinations of IC₅₀ made by whole cell patch clampelectrophysiology with HEK 293 as described in Example 36 herein above.The sustained IC₅₀ was derived from the current 400 msecs after voltageramp from −80 mV to +30 mV. Pool #2 samples comprised di-PEGylatedpeptibodies and Pool #3 samples comprised mono-PEGylated peptibodies.

PEGylated Peptibody Pool # IC50 Sustained (nM) PEG-Fc-L10-SHK(2-35) 30.175(n = 4) PEG-Fc-L10-SHK(2-35) 2 0.158(n = 4) PEG-Fc-L10-OSK1 30.256(n = 3) PEG-Fc-L10-OSK1 2 0.332(n = 3)

Example 48 PEGylated Toxin Peptides

Shk and Osk-1 PEGylation, purification and analysis. Synthetic Shk orOSK1-1 toxin peptides were selectively PEGylated by reductive alkylationat their N-termini. Conjugation was achieved, with either Shk or OSK-1toxin peptides, at 2 mg/ml in 50 mM NaH₂PO₄, pH 4.5 reaction buffercontaining 20 mM sodium cyanoborohydride and a 2 molar excess of 20 kDamonomethoxy-PEG-aldehyde (Nektar Therapeutics, Huntsville, Ala.).Conjugation reactions were stirred overnight at room temperature, andtheir progress was monitored by RP-HPLC. Completed reactions werequenched by 4-fold dilution with 20 mM NaOAc, pH 4, adjusted to pH 3.5and chilled to 4° C. The PEG-peptides were then purifiedchromatographically at 4° C.; using SP Sepharose HP columns (GEHealthcare, Piscataway, N.J.) eluted with linear 0-1M NaCl gradients in20 mM NaOAc, pH 4.0. (FIG. 68A and FIG. 68B) Eluted peak fractions wereanalyzed by SDS-PAGE and RP-HPLC and pooling determined by purity >97%.Principle contaminants observed were di-PEGylated toxin peptide andunmodified toxin peptide. Selected pools were concentrated to 2-5 mg/mlby centrifugal filtration against 3 kDa MWCO membranes and dialyzed into10 mM NaOAc, pH 4 with 5% sorbitol. Dialyzed pools were then sterilefiltered through 0.2 micron filters and purity determined to be >97% bySDS-PAGE and RP-HPLC (FIG. 69A and FIG. 69B). Reverse-phase HPLC wasperformed on an Agilent 1100 model HPLC running a Zorbax 5 μm 300SB-C84.6×50 mm column (Phenomenex) in 0.1% TFA/H₂0 at 1 ml/min and columntemperature maintained at 40° C. Samples of PEG-peptide (20 μg) wereinjected and eluted in a linear 6-60% gradient while monitoringwavelengths 215 nm and 280 nm.

Electrophysiology performed by patch clamp on whole cells (see, Example36) yielded a peak IC50 of 1.285 nM for PEG-OSK1 and 0.169 nM forPEG-ShK[1-35] (FIG. 74), in a concentration dependent block of theoutward potassium current recorded from HEK293 cells stably expressinghuman Kv1.3 channel. The purified PEG-ShK[1-35] molecule, also referredto as “20K PEG-ShK[1-35]” and “PEG-ShK”, had a much longer half-life invivo than the small ShK peptide (FIG. 59 and FIG. 60). PEG-ShK[1-35]suppressed severe autoimmune encephalomyelitis in rats (Example 45,FIGS. 61-63) and showed greater efficacy than the small native ShKpeptide.

PEG conjugates of OSK1 peptide analogs were also generated and testedfor activity in blocking T cell inflammation in the human whole bloodassay (Example 46). As shown in Table 43, OSK1[Ala12], OSK1[Ala29],OSK1[Nal34] and OSK1[Ala29, 1Nal34] analogs containing an N-terminal 20KPEG conjugate, all provided potent blockade of the whole blood cytokineresponse in this assay. The 20K PEG-ShK was also highly active (Table43).

Example 49 Fc Loop Insertions of ShK and OSK1 Toxin Peptides

As exemplified in FIG. 70, FIG. 71, FIG. 72, and FIG. 73,disulphide-constrained toxin peptides were inserted into the human IgG1Fc-loop domain, defined as the sequence D₁₃₇E₁₃₈T₁₃₉T₁₄₀K₁₄₁, accordingto the method published in Example 1 in Gegg et al., Modified Fcmolecules, WO 2006/036834 A2 [PCT/US2005/034273]). ExemplaryFcLoop-L2-OsK1-L2, FcLoop-L2-ShK-L2, FcLoop-L2-ShK-L4, andFcLoop-L4-OsK1-L2 were made having three linked domains. These werecollected, purified and submitted for functional testing.

The peptide insertion for these examples was between Fc residues Leu₁₃₉and Thr₁₄₀ and included 2-4 Gly residues as linkers flanking either sideof the inserted peptide. However, alternate insertion sites for thehuman IgG1 Fc sequence, or different linkers, are also useful in thepractice of the present invention, as is known in the art, e.g., asdescribed in Example 13 of Gegg et al., Modified Fc molecules, WO2006/036834 A2 [PCT/US2005/034273]).

Purified FcLoop OSK1 and FcLoop ShK1 molecules were tested in the wholeblood assay of inflammation (see, Example 46). FcLoop-L2-OsK1-L2,FcLoop-L4-OsK1-L4 and FcLoop-L2-ShK-L2 toxin conjugates all providedpotent blockade of the whole blood cytokine response in this assay, withIC50 values in the pM range (Table 43).

Example 50 Purification of ShK(2-35)-L-Fc from E. coli

Frozen, E. coli paste (117 g), obtained as described in Example 16herein above, was combined with 1200 ml of room temperature 50 mM trisHCl, 5 mM EDTA, pH 7.5 and was brought to about 0.1 mg/ml hen egg whitelysozyme. The suspended paste was passed through a chilledmicrofluidizer twice at 12,000 PSI. The cell lysate was then centrifugedat 17,700 g for 30 min at 4° C. The pellet was then resuspended in 1200ml 1% deoxycholic acid using a tissue grinder and then centrifuged at17,700 g for 30 min at 4° C. The pellet was then resuspended in 1200 mlwater using a tissue grinder and then centrifuged at 17,700 g for 30 minat 4° C. 6.4 g of the pellet (total 14.2 g) was then dissolved in 128 ml8 M guanidine HCl, 50 mM tris HCl, pH 8.0. 120 ml of the pellet solutionwas then incubated with 0.67 ml of 1 M DTT for 60 min at 37° C. Thereduced material was transferred to 5500 ml of the refolding buffer (3 Murea, 50 mM tris, 160 mM arginine HCl, 2.5 mM EDTA, 2.5 mM cystamineHCl, 4 mM cysteine, pH 9.5) at 2 ml/min, 4° C. with vigorous stirring.The stirring rate was then slowed and the incubation was continued for 3days at 4° C.

The refold was diluted with 5.5 L of water, and the pH was adjusted to8.0 using acetic acid, then the solution was filtered through a 0.22 μmcellulose acetate filter and loaded on to a 35 ml Amersham QSepharose-FF (2.6 cm I.D.) column at 10 ml/min in Q-Buffer A (20 mMTris, pH 8.5) at 8° C. with an inline 35 ml Amersham Mab Select column(2.6 cm I.D.). After loading, the Q Sepharose column was removed fromthe circuit, and the remaining chromatography was carried out on the MabSelect column. The column was washed with several column volumes ofQ-Buffer A, followed by elution using a step to 100 mM glycine pH 3.0.The fractions containing the desired product immediately loaded on to a5.0 ml Amersham SP-Sepharose HP column at 5.0 ml/min in S-Buffer A (10mM NaH₂PO₄, pH 7.0) at 8° C. The column was then washed with severalcolumn volumes of S-Buffer A followed by a linear gradient from 5% to60% S-Buffer B (10 mM NaH₂PO₄, 1 M NaCl, pH 7.0) followed by a step to100% S-Buffer B. Fractions were then analyzed using a Coomassiebrilliant blue stained tris-glycine 4-20% SDS-PAGE. The fractionscontaining the bulk of the desired product were pooled and then appliedto a 50 ml MEP Hypercel column (2.6 cm I.D.) at 10 ml/min in MEP BufferA (20 mM tris, 200 mM NaCl, pH 8.0) at 8° C. Column was eluted with alinear gradient from 5% to 50% MEP Buffer B (50 mM sodium citrate pH4.0) followed by a step to 100% MEP Buffer B. Fractions were thenanalyzed using a Coomassie brilliant blue stained tris-glycine 4-20%SDS-PAGE, and the fractions containing the bulk of the desired productwere pooled.

The MEP-pool was then concentrated to about 10 ml using a Pall Jumbo-Sepwith a 10 kDa membrane. A spectral scan was then conducted on 50 μl ofthe combined pool diluted in 700 μl PBS using a Hewlett Packard 8453spectrophotometer (FIG. 76A). Then concentration of the material wasdetermined to be 3.7 mg/ml using a calculated molecular mass of 30,253and extinction coefficient of 36,900 M⁻¹ cm⁻¹. The purity of thematerial was then assessed using a Coomassie brilliant blue stainedtris-glycine 4-20% SDS-PAGE (FIG. 76B). The macromolecular state of theproduct was then determined using size exclusion chromatography on 70 μgof the product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm (FIG. 76C). The product was then subject tomass spectral analysis by chromatographing approximately 4 μg of thesample through a RP-HPLC column (Vydac C₄, 1×150 mm). Solvent A was 0.1%trifluoroacetic acid in water and solvent B was 0.1% trifluoroaceticacid in 90% acetonitrile, 10% water. The column was pre-equilibrated in10% solvent B at a flow rate of 80 μl per min. The protein was elutedusing a linear gradient of 10% to 90% solvent B over 30 min. Part of theeffluent was directed into a LCQ ion trap mass spectrometer. The massspectrum was deconvoluted using the Bioworks software provided by themass spectrometer manufacturer. (FIG. 76D). The product was filteredthrough a 0.22 μm cellulose acetate filter and then stored at −80° C.

In Table 35, IC50 data for the purified E. coli-derived ShK[2-35]-L-Fcare compared to some other embodiments of the inventive composition ofmatter.

TABLE 35 E. coli-derived recombinant Fc-L-ShK[1-35], Fc-L-ShK[2-35],Fc-L-OSK1, Shk[1-35]-L- Fc and ShK[2-35]-L-Fc peptibodies containing Fcat either the N-terminus or C-terminus show potent blockade of humanKv1.3. The activity of the CHO-derived Fc-L10-ShK[1-35] R1Q mutant isalso shown. Whole cell patch clamp electrophysiology (WCVC), by methodsdescribed in Example 36, was performed using HEK293/Kvl.3 cells and theIC50 shown is the average from dose-response curves from 3 or morecells. IonWorks ™ (IWQ) planar patch clamp electrophysiology by methodsdescribed in Example 44 was on CHO/Kv1.3 cells and the average IC50 isshown. The inventive molecules were obtained by methods as described inthe indicated Example: E. coli-derived Fc-L-ShK[1-35] (Example 3 andExample 38), E. coli-derived Fc-L-ShK[2-35] (Example 4 and Example 39),E. coli Fc-L-OSK1 (Example 10 and Example 40), ShK[1-35]-L-35 Fc(Example 15 and Example 51), and ShK[2-35]-L-Fc (Example 16 and thisExample 50). CHO-derived Fc-L10-ShK[1-35] R1Q molecule was generatedusing methods similar to those described for CHO-derivedFc-L10-ShK[1-35]. Kv1.3 IC₅₀ Kv1.3 IC₅₀ Molecule by WCVC (nM) by IWQ(nM) E. coli-derived Fc-L-ShK[1-35] 1.4 E. coli-derived Fc-L-ShK[2-35]1.3 2.8 E. coli-derived Fc-L-OSK1 3.2 E. coli-derived Shk[1-35]-L-Fc 2.4E. coli-derived ShK[2-35]-L-Fc 4.9 CHO-derived Fc-L10-ShK[1-35] 2.2 R1Q

Example 51 Purification of Met-ShK(1-35)-Fc from E. coli

Frozen, E. coli paste (65 g), obtained as described in Example 15 hereinabove was combined with 660 ml of room temperature 50 mM tris HCl, 5 mMEDTA, pH 7.5 and was brought to about 0.1 mg/ml hen egg white lysozyme.The suspended paste was passed through a chilled microfluidizer twice at12,000 PSI. The cell lysate was then centrifuged at 17,700 g for 30 minat 4° C. The pellet was then resuspended in 660 ml 1% deoxycholic acidusing a tissue grinder and then centrifuged at 17,700 g for 30 min at 4°C. The pellet was then resuspended in 660 ml water using a tissuegrinder and then centrifuged at 17,700 g for 30 min at 4° C. 13 g of thepellet was then dissolved in 130 ml 8 M guanidine HCl, 50 mM tris HCl,pH 8.0. 10 ml of the pellet solution was then incubated with 0.1 ml of 1M DTT for 60 min at 37° C. The reduced material was transferred to 1000ml of the refolding buffer (2 M urea, 50 mM tris, 160 mM arginine HCl,2.5 mM EDTA, 1.2 mM cystamine HCl, 4 mM cysteine, pH 8.5) at 2 ml/min,4° C. with vigorous stirring. The stirring rate was then slowed and theincubation was continued for 3 days at 4° C.

The refold was diluted with 1 L of water, and filtered through a 0.22 μmcellulose acetate filter then loaded on to a 35 ml Amersham QSepharose-FF (2.6 cm I.D.) column at 10 ml/min in Q-Buffer A (20 mMTris, pH 8.5) at 8° C. with an inline 35 ml Amersham Mab Select column(2.6 cm I.D.). After loading, the Q Sepharose column was removed fromthe circuit, and the remaining chromatography was carried out on the MabSelect column. The column was washed with several column volumes ofQ-Buffer A, followed by elution using a step to 100 mM glycine pH 3.0.The fractions containing the desired product immediately loaded on to a5.0 ml Amersham SP-Sepharose HP column at 5.0 ml/min in S-Buffer A (20mM NaH₂PO₄, pH 7.0) at 8° C. The column was then washed with severalcolumn volumes of S-Buffer A followed by a linear gradient from 5% to60% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.0) followed by a step to100% S-Buffer B. Fractions were then analyzed using a Coomassiebrilliant blue stained tris-glycine 4-20% SDS-PAGE. The fractionscontaining the bulk of the desired product were pooled.

The S-pool was then concentrated to about 10 ml using a Pall Jumbo-Sepwith a 10 kDa membrane. A spectral scan was then conducted on 20 μl ofthe combined pool diluted in 700 μl PBS using a Hewlett Packard 8453spectrophotometer (FIG. 77A). Then concentration of the material wasdetermined to be 3.1 mg/ml using a calculated molecular mass of 30,409and extinction coefficient of 36,900 M⁻¹ cm⁻¹. The purity of thematerial was then assessed using a Coomassie brilliant blue stainedtris-glycine 4-20% SDS-PAGE (FIG. 77B). The macromolecular state of theproduct was then determined using size exclusion chromatography on 93 μgof the product injected on to a Phenomenex BioSep SEC 3000 column(7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observingthe absorbance at 280 nm (FIG. 77C). The product was then subject tomass spectral analysis by MALDI mass spectrometry.

An aliquot of the sample was spotted with the MALDI matrix sinapinicacid on sample plate. A Voyager DE-RP time-of-flight mass spectrometerequipped with a nitrogen laser (337 nm, 3 ns pulse) was used to collectspectra. The positive ion/linear mode was used, with an acceleratingvoltage of 25 kV. Each spectrum was produced by accumulating data from˜200 laser shots (FIG. 77D). External mass calibration was accomplishedusing purified proteins of known molecular masses.

The IC50 for blockade of human Kv1.3 by purified E. coli-derivedMet-ShK(1-35)-Fc, also referred to as “ShK[1-35]-L-Fc”, is shown inTable 35 herein above.

Example 52 Bacterial Expression of OsK1-L-Fc Inhibitor of Kv1.3

The methods to clone and express the peptibody in bacteria were asdescribed in Example 3. The vector used was pAMG21amgR-pep-Fc and theoligos listed below were used to generate a duplex (see below) forcloning and expression in bacteria of OsK1-L-Fc. Oligos used to formduplex are shown below:

//SEQ ID NO: 1347 GGGTGTTATCATCAACGTTAAATGCAAAATCTCCCGTCAGTGCCTGGAACCGTGCAAAAAAGCTGGTATGCGT; //SEQ ID NO: 1348TTCGGTAAATGCATGAACGGTAAATGCCACTGCACCCCGAAATCTGGTGG TGGTGGTTCT;//SEQ ID NO: 1349 CACCAGAACCACCACCACCACCAGATTTCGGGGTGCAGTGGCATTTACCGTTCATGCATTTACCGAAACGCAT; //SEQ ID NO: 1310ACCAGCTTTTTTGCACGGTTCCAGGCACTGACGGGAGATTTTGCATTTAA CGTTGATGATAAC;

The oligos shown above were used to form the duplex shown below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 53 Bacterial Expression of Gly-ShK(1-35)-L-Fc Inhibitor of Kv1.3

The methods to clone and express the peptibody in bacteria were asdescribed in Example 3. The vector used was pAMG21amgR-pep-Fc and theoligos listed below were used to generate a duplex (see below) forcloning and expression in bacteria of Gly-ShK(1-35)-L-Fc. Oligos used toform duplex are shown below:

//SEQ ID NO: 1313 GGGTCGTTCTTGTATTGATACTATTCCAAAATCTCGTTGTACTGCTTTTCAATGTAAACATTCTATGAAATATCGTCTTTCTT; //SEQ ID NO: 1314TTTGTCGTAAAACTTGTGGTACTTGTTCTGGTGGTGGTGGTTCT; //SEQ ID NO: 1353CACCAGAACCACCACCACCAGAACAAGTACCACAAGTTTTACGACAAAAAGAAAGACGATATTTCATAGAATGTTTACATTGA; //SEQ ID NO: 1354AAAGCAGTACAACGAGATTTTGGAATAGTATCAATACAAGAACG

The oligos shown above were used to form the duplex shown below:

Bacterial expression of the peptibody was as described in Example 3 andpaste was stored frozen.

Example 54 Bacterial Expression of CH2-OSK1 Inhibitor of Kv1.3

The methods to clone and express the fusion of a CH2 domain of an Fcwith OSK1 in bacteria were generally as described in Example 3. Thevector used was pAMG21.G2.H6.G3.CH2.(G4S)2.OSK. Briefly, the pAMG21vector was modified to remove the multi-cloning site's BamHI. Thisallowed the BamHI in front of the OSK as a site to swap out differentsequences for fusion with the OSK. The sequence upstream of the OSK1coding sequence was ligated between the NdeI and BamHI sites.

The sequence of the entire vector, including the insert was thefollowing:

//SEQ ID NO: 4914 gtcgtcaacgaccccccattcaagaacagcaagcagcattgagaactttggaatccagtccctcttccacctgctgaccggatcagcagtccccggaacatcgtagctgacgccttcgcgttgctcagttgtccaaccccggaaacgggaaaaagcaagttttccccgctcccggcgtttcaataactgaaaaccatactatttcacagtttaaatcacattaaacgacagtaatccccgttgatttgtgcgccaacacagatcttcgtcacaattctcaagtcgctgatttcaaaaaactgtagtatcctctgcgaaacgatccctgtttgagtattgaggaggcgagatgtcgcagacagaaaatgcagtgacttcctcattgagtcaaaagcggtttgtgcgcagaggtaagcctatgactgactctgagaaacaaatggccgttgttgcaagaaaacgtcttacacacaaagagataaaagtttttgtcaaaaatcctctgaaggatctcatggttgagtactgcgagagagaggggataacacaggctcagttcgttgagaaaatcatcaaagatgaactgcaaagactggatatactaaagtaaagactttactttgtggcgtagcatgctagattactgatcgtttaaggaattttgtggctggccacgccgtaaggtggcaaggaactggttctgatgtggatttacaggagccagaaaagcaaaaaccccgataatcttcttcaacttttgcgagtacgaaaagattaccggggcccacttaaaccgtatagccaacaattcagctatgcggggagtatagttatatgcccggaaaagttcaagacttctttctgtgctcgctccttctgcgcattgtaagtgcaggatggtgtgactgatcttcaccaaacgtattaccgccaggtaaagaacccgaatccggtgtttacaccccgtgaaggtgcaggaacgctgaagttctgcgaaaaactgatggaaaaggcggtgggcttcacttcccgttttgatttcgccattcatgtggcgcacgcccgttcgcgtgatctgcgtcgccgtatgccaccagtgctgcgtcgtcgggctattgatgcgctcttgcaggggctgtgtttccactatgacccgctggccaaccgcgtccagtgctccatcaccacgctggccattgagtgcggactggcgacggagtctgctgccggaaaactctccatcacccgtgccacccgtgccctgacgttcctgtcagagctgggactgattacctaccagacggaatatgacccgcttatcgggtgctacattccgaccgatatcacgttcacatctgcactgtttgctgccctcgatgtatcagaggaggcagtggccgccgcgcgccgcagccgtgtggtatgggaaaacaaacaacgcaaaaagcaggggctggataccctgggcatggatgaactgatagcgaaagcctggcgttttgttcgtgagcgttttcgcagttatcagacagagcttaagtcccgtggaataaagcgtgcccgtgcgcgtcgtgatgcggacagggaacgtcaggatattgtcaccctggtgaaacggcagctgacgcgcgaaatcgcggaagggcgcttcactgccaatcgtgaggcggtaaaacgcgaagttgagcgtcgtgtgaaggagcgcatgattctgtcacgtaaccgtaattacagccggctggccacagcttccccctgaaagtgacctcctctgaataatccggcctgcgccggaggcttccgcacgtctgaagcccgacagcgcacaaaaaatcagcaccacatacaaaaaacaacctcatcatccagcttctggtgcatccggccccccctgttttcgatacaaaacacgcctcacagacggggaattttgcttatccacattaaactgcaagggacttccccataaggttacaaccgttcatgtcataaagcgccatccgccagcgttacagggtgcaatgtatcttttaaacacctgtttatatctcctttaaactacttaattacattcatttaaaaagaaaacctattcactgcctgtccttggacagacagatatgcacctcccaccgcaagcggcgggcccctaccggagccgctttagttacaacactcagacacaaccaccagaaaaaccccggtccagcgcagaactgaaaccacaaagcccctccctcataactgaaaagcggccccgccccggtccgaagggccggaacagagtcgcttttaattatgaatgttgtaactacttcatcatcgctgtcagtcttctcgctggaagttctcagtacacgctcgtaagcggccctgacggcccgctaacgcggagatacgccccgacttcgggtaaaccctcgtcgggaccactccgaccgcgcacagaagctctctcatggctgaaagcgggtatggtctggcagggctggggatgggtaaggtgaaatctatcaatcagtaccggcttacgccgggcttcggcggttttactcctgtttcatatatgaaacaacaggtcaccgccttccatgccgctgatgcggcatatcctggtaacgatatctgaattgttatacatgtgtatatacgtggtaatgacaaaaataggacaagttaaaaatttacaggcgatgcaatgattcaaacacgtaatcaatatcgggggtgggcgaagaactccagcatgagatccccgcgctggaggatcatccagccggcgtcccggaaaacgattccgaagcccaacctttcatagaaggcggcggtggaatcgaaatctcgtgatggcaggttgggcgtcgcttggtcggtcatttcgaaccccagagtcccgctcagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagcggcgataccgtaaagcacgaggaagcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacgctatgtcctgatagcggtccgccacacccagccggccacagtcgatgaatccagaaaagcggccattttccaccatgatattcggcaagcaggcatcgccatgagtcacgacgagatcctcgccgtcgggcatgcgcgccttgagcctggcgaacagttcggctggcgcgagcccctgatgctcttcgtccagatcatcctgatcgacaagaccggcttccatccgagtacgtgctcgctcgatgcgatgtttcgcttggtggtcgaatgggcaggtagccggatcaagcgtatgcagccgccgcattgcatcagccatgatggatactttctcggcaggagcaaggtgagatgacaggagatcctgccccggcacttcgcccaatagcagccagtcccttcccgcttcagtgacaacgtcgagcacagctgcgcaaggaacgcccgtcgtggccagccacgatagccgcgctgcctcgtcctgcaattcattcaggacaccggacaggtcggtcttgacaaaaagaaccgggcgcccctgcgctgacagccggaacacggcggcatcagagcagccgattgtctgttgtgcccagtcatagccgaatagcctctccacccaagcggccggagaacctgcgtgcaatccatcttgttcaatcatgcgaaacgatcctcatcctgtctcttgatctgatcttgatcccctgcgccatcagatccttggcggcaagaaagccatccagtttactttgcagggcttcccaaccttaccagagggcgccccagctggcaattccggttcgcttgctgtccataaaaccgcccagtctagctatcgccatgtaagcccactgcaagctacctgctttctctttgcgcttgcgttttcccttgtccagatagcccagtagctgacattcatccggggtcagcaccgtttctgcggactggctttctacgtgttccgcttcctttagcagcccttgcgccctgagtgcttgcggcagcgtgaagctacatatatgtgatccgggcaaatcgctgaatattccttttgtctccgaccatcaggcacctgagtcgctgtctttttcgtgacattcagttcgctgcgctcacggctctggcagtgaatgggggtaaatggcactacaggcgccttttatggattcatgcaaggaaactacccataatacaagaaaagcccgtcacgggcttctcagggcgttttatggcgggtctgctatgtggtgctatctgactttttgctgttcagcagttcctgccctctgattttccagtctgaccacttcggattatcccgtgacaggtcattcagactggctaatgcacccagtaaggcagcggtatcatcaacaggcttacccgtcttactgtcgaagacgtgcgtaacgtatgcatggtctccccatgcgagagtagggaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgtttctacaaactcttttgtttatttttctaaatacattcaaatatggacgtcgtacttaacttttaaagtatgggcaatcaattgctcctgttaaaattgctttagaaatactttggcagcggtttgttgtattgagtttcatttgcgcattggttaaatggaaagtgaccgtgcgcttactacagcctaatatttttgaaatatcccaagagctttttccttcgcatgcccacgctaaacattctttttctcttttggttaaatcgttgtttgatttattatttgctatatttatttttcgataattatcaactagagaaggaacaattaatggtatgttcatacacgcatgtaaaaataaactatctatatagttgtctttctctgaatgtgcaaaactaagcattccgaagccattattagcagtatgaatagggaaactaaacccagtgataagacctgatgatttcgcttctttaattacatttggagattttttatttacagcattgttttcaaatatattccaattaatcggtgaatgattggagttagaataatctactataggatcatattttattaaattagcgtcatcataatattgcctccattttttagggtaattatccagaattgaaatatcagatttaaccatagaatgaggataaatgatcgcgagtaaataatattcacaatgtaccattttagtcatatcagataagcattgattaatatcattattgcttctacaggctttaattttattaattattctgtaagtgtcgtcggcatttatgtctttcatacccatctctttatccttacctattgtttgtcgcaagttttgcgtgttatatatcattaaaacggtaatagattgacatttgattctaataaattggatttttgtcacactattatatcgcttgaaatacaattgtttaacataagtacctgtaggatcgtacaggtttacgcaagaaaatggtttgttatagtcgattaatcgatttgattctagatttgttttaactaattaaaggaggaataacatatgggcggccatcatcatcatcatcatggcgggggaccgtcagttttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccggcggcggcggcagcggcggcggcggatccggtgttatcatcaacgttaaatgcaaaatctcccgtcagtgcctggaaccgtgcaaaaaagctggtatgcgtttcggtaaatgcatgaacggtaaatgccactgcaccccgaaataatgaattcgagctcactagtgtcgacctgcagggtaccatggaagcttactcgaagatccgcggaaagaagaagaagaagaagaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaaccgctcttcacgctcttcacgcggataaataagtaacgatccggtccagtaatgacctcagaactccatctggatttgttcagaacgctcggttgccgccgggcgttttttattggtgagaatcgcagcaacttg tcgcgccaatcgagccatgtc.

The insert DNA sequence was the following:

//SEQ ID NO: 4915 atgggcggccatcatcatcatcatcatggcgggggaccgtcagttttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccggcggcggcggcagcggcggcggcggatccggtgttatcatcaacgttaaatgcaaaatctcccgtcagtgcctggaaccgtgcaaaaaagctggtatgcgtttcggtaaatgcatgaacggtaaatgccactgcaccccgaaa.

The amino acid sequence of the CH2-OSK1 fusion protein product was thefollowing:

//SEQ ID NO: 4917 MGGHHHHHHGGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISGGGGSGGGGSGVIINVKVKISRQCLEPCKKAGMRFGKC MNGKCHCTPK.

SEQ ID NO:4917 includes the OSK1 sequence

(SEQ ID NO: 25) GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK.

Purification and refolding of CH2-OSK1 expressed in bacteria. Frozen, E.coli paste (13.8 g) was combined with 180 ml of room temperature 50 mMtris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen eggwhite lysozyme. The suspended paste was passed through a chilledmicrofluidizer twice at 12,000 PSI. The cell lysate was then centrifugedat 17,700 g for 50 min at 4° C. The pellet was then resuspended in 90 ml1% deoxycholate using a tissue grinder and then centrifuged at 15,300 gfor 40 min at 4° C. The pellet was then resuspended in 90 ml water usinga tissue grinder and then centrifuged at 15,300 g for 40 min at 4° C.The pellet (3.2 g) was then dissolved in 64 ml 8 M guanidine HCl, 50 mMtris HCl, pH 8.0. The suspension was then incubated at room temperature(about 23° C.) for 30 min with gentle agitation followed bycentrifugation at 15,300 g for 30 min at 4° C. The supernatant (22 ml)was then reduced by adding 220 μl 1 M dithiothreitol and incubating at37° C. for 30 minutes. The reduced suspension (20 ml) was transferred to2000 ml of the refolding buffer (1 M urea, 50 mM ethanolamine, 160 mMarginine HCl, 0.02% NaN₃, 1.2 mM cystamine HCl, 4 mM cysteine, pH 9.8)at 4° C. with vigorous stirring. The stirring rate was then slowed andthe incubation was continued for approximately 2.5 days at 4° C.

Ten milliliters of 500 mM imidazole was added to the refolding solutionand the pH was adjusted to pH to 8.0 with 5 M acetic acid. The refoldwas then filtered through a 0.45 μm cellulose acetate filter with twopre-filters. This material was then loaded on to a 50 ml Qiagen Ni-NTASuperflow column (2.6 cm ID) in Ni-Buffer A (50 mM NaH₂PO₄, 300 mM NaCl,pH 7.5) at 15 ml/min 13° C. The column was then washed with 10 columnvolumes of Ni-Buffer A followed by 8% Ni-Buffer B (250 mM imidazole, 50mM NaH₂PO₄, 300 mM NaCl, pH 7.5) at 25 ml/min. The column was theneluted with 60% Ni-Buffer B followed by 100% Ni-Buffer B at 10 ml/min.The peak fractions were collected and dialyzed against S-Buffer A (10 mMNaH₂PO₄, pH 7.1)

The dialyzed sample was then loaded on to a 5 ml Amersham SP-HP HiTrapcolumn at 5 ml/min in S-Buffer A at 13° C. The column was then washedwith several column volumes of S-Buffer A, followed by elution with alinear gradient from 0% to 60% S-Buffer B (10 mM NaH₂PO₄, 1 M NaCl, pH7.1) followed by a step to 100% S-Buffer B at 1.5 ml/min 13° C.Fractions were then analyzed using a Coomassie brilliant blue stainedtris-glycine 4-20% SDS-PAGE, and the fractions containing the desiredproduct were pooled based on these data. The pool was then concentratedto about 1.6 ml using a Pall Macrosep with a 10 kDa membrane at 4° C.The concentrated sample was then filtered through a 0.22 μm celluloseacetate centrifugal filter.

A spectral scan was then conducted on 10 μl of the combined pool dilutedin 150 μl water using a Hewlett Packard 8453 spectrophotometer (FIG.78). The concentration of the filtered material was determined to be3.35 mg/ml using a calculated molecular mass of 17,373 g/mol andextinction coefficient of 17,460 M⁻¹ cm⁻¹. The purity of the filteredmaterial was then assessed using a Coomassie brilliant blue stainedtris-glycine 4-20% SDS-PAGE (FIG. 79). The endotoxin level was thendetermined using a Charles River Laboratories Endosafe-PTS system(0.05-5 EU/ml sensitivity) using a 67-fold dilution of the sample inCharles Rivers Endotoxin Specific Buffer BG120 yielding a result of <1EU/mg protein. The macromolecular state of the product was thendetermined using size exclusion chromatography on 50 μg of the productinjected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mMNaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280nm (FIG. 80). The product was then subject to mass spectral analysis bychromatographing approximately 4 μg of the sample through a RP-HPLCcolumn (Vydac C₄, 1×150 mm). Solvent A was 0.1% trifluoroacetic acid inwater and solvent B was 0.1% trifluoroacetic acid in 90% acetonitrile,10% water. The column was pre-equilibrated in 10% solvent B at a flowrate of 80 μl per min. The protein was eluted using a linear gradient of10% to 90% solvent B over 30 min. Part of the effluent was directed intoa LCQ ion trap mass spectrometer. The mass spectrum was deconvolutedusing the Bioworks software provided by the mass spectrometermanufacturer. (FIG. 81). The product was then stored at −80° C.

PEGylation of CH2-OSK1. The CH2-OSK1 fusion protein was diluted to 2mg/ml in 50 mM sodium acetate, 10 mM sodium cyanoborohydride, pH 4.8with a 4-fold molar excess of 20 kD methoxy-PEG-aldehyde (NektarTherapeutics, Huntsville, Ala.). The reaction was allowed to proceedovernight (˜18 hrs) at 4° C. Upon completion, reaction was quenched with4 volumes of 10 mM sodium acetate, 50 mM NaCl, pH 5, then loaded at 0.7mg protein/ml resin to an SP Sepharose HP column (GE Healthcare,Piscataway, N.J.) equilibrated in 10 mM sodium acetate, 50 mM NaCl, pH5. The mono-PEGylated CH2-Osk fusion was eluted with a linear 50 mM-1 MNaCl gradient (FIG. 82). Peak fractions were evaluated by SDS-PAGE andthe mono-PEG-CH2-OSK1 fractions pooled, concentrated and dialyzed intoDulbecco's Phosphate Buffered Saline. The final product was analyzed bySDS-PAGE (FIG. 83).

As shown in Table 43, the purified CH2-OSK1(“L2-6H-L3-CH2-L10-OsK1(1-38)”) and PEGylated CH2-OSK1 (“20 kPEG-L2-6H-L3-CH2-L10-OSK1(1-38)”) molecules were active in blockinginflammation in the human whole blood assay (See, Example 46).

Example 55 Bioactivity of OSK1 Peptide Analogs

The activity of OSK1 peptide analogs in blocking human Kv1.3 versushuman Kv1.1 current is shown in FIGS. 84 through 86 and Tables 37-41.Three electrophysiology techniques were used (See, e.g., Example 36 andExample 44). Whole cell patch clamp (FIGS. 84 & 85 and Table 41)represents a low throughput technique which is well established in thefield and has been available for many years. We also used two new planarpatch clamp techniques, PatchXpress and IonWorks Quattro, with improvedthroughput which facilitate assessment of potency and selectivity ofnovel OSK1 analogs described in this application. The PatchXpresstechnique is of moderate throughput and the novel OSK1[Ala-12] analog(SEQ ID No:1410) had similar Kv1.3 potency and selectivity over Kv1.1 tothat observed by whole cell patch clamp (FIG. 84 and Table 41). IonWorksQuattro represents a 384-well planar patch clamp electrophysiologysystem of high throughput. Using this IonWorks system, the novelOSK1[Ala-29] analog (SEQ ID No:1424) showed potent inhibition of theKv1.3 current and improved selectivity over Kv1.1 (FIG. 86). TheOSK1[Ala-29] analog (SEQ ID No:1424) showed similar Kv1.3 activity andselectivity over Kv1.1 by whole cell patch clamp electrophysiology (FIG.85) to that observed by IonWorks (FIG. 86). The Kv1.3 and Kv1.1activities of Alanine, Arginine, Glutamic acid and 1-Naphthylalanineanalogs of OSK1 were determined by IonWorks electrophysiology and isreported in Tables 37-40. OSK1 peptide analogs identified by IonWorks tohave good potency or Kv1.3 selectivity, were tested further in wholecell patch clamp studies (see, Table 41). The Kv1.3 IC50 of the His34Alaanalog of OSK1 (SEQ ID No:1428) was 797 fold lower than its IC50 againstKv1.1 (Table 41), demonstrating that this analog is a highly selectiveKv1.3 inhibitor. In this same assay, native OSK1 (SEQ ID NO: 25) showedonly slight Kv1.3 selectivity, with the Kv1.1 IC50 being only 5 foldhigher than Kv1.3.

The novel OSK1 peptide analogs described in this application whichinhibit Kv1.3 are useful in the treatment of autoimmune disease andinflammation. Kv1.3 is expressed on T cells and Kv1.3 inhibitorssuppress inflammation by these cells. As one measure of inflammationmediated by T cells, we examined the impact of OSK1 analogs on IL-2 andIFN-g production in human whole blood following addition of apro-inflammatory stimulus (Tables 36-40 and Table 42). “WB/IL-2” inthese tables refers to the assay measuring IL-2 response of whole blood(see, Example 46), whereas “WB/IFNg” refers to the assay measuring IFNgresponse of whole blood (see, Example 46). The IC50 values listed inTables 36-40 and 42, represent the average IC50 value determined fromexperiments done with two or more blood donors. The whole blood assay(see Example 46) allows for a combined measurement of the potency of theanalogs in blocking inflammation and Kv1.3, as well as an assessment ofthe stability of the molecules in a complex biological fluid. Using thisassay, several OSK1 analogs were examined and found to potently suppressinflammation (FIGS. 90C & 90D, Tables 36-40). Some of these analogsshowed reduced activity in this whole blood assay, which may indicatethat these residues play an important role in binding Kv1.3. Relative tothe immunosuppressive agent cyclosporin A, Kv1.3 peptide inhibitorsShK-Ala22, OSK1-Ala29, and OSK1-Ala12 were several orders of magnitudemore potent in blocking the cytokine response in human whole blood (FIG.90).

The solution NMR structure of OSK1 has been solved and is provided aspdb accession number “1SCO” in Entrez's Molecular Modeling Database(MMDB) [J. Chen et al. (2003) Nucleic Acids Res. 31, 474-7]. FIG. 89shows space filling (FIG. 89A, 89B, 89D) and worm (FIG. 89C) Cn3Drendering of the OSK1 structure. Light colored OSK1 amino acid residuesPhe25, Gly26, Lys27, Met29 and Asn30 are shown in FIG. 89B. Some analogsof these residues were found to significantly reduce Kv1.3 activity(Tables 37-40), implying that these residues may make important contactswith the Kv1.3 channel. The molecular structure shown in FIG. 89Aindicates these amino acids reside on a common surface of the OSK1three-dimensional (3D) structure. FIG. 89D shows OSK1 residues (lightshading) Ser11, Met29 and His34. These residues when converted to someamino acid analogs, provide improved Kv1.3 selectivity over Kv1.1 (Table41). Although about 23 amino acids are between residues Ser11 and His34in the contiguous polypeptide chain, the structure shown in FIG. 89Dillustrates that in the 3D structure of the folded molecule theseresidues are relatively close to one another. Upon comparing FIGS. 89Dand 89B, one can see that residues His34 and Ser11 (FIG. 89D) are on theleft and right side, respectively, and adjacent to the major Kv1.3contact surface displayed in FIG. 89B. It is envisioned that molecularmodeling can be used to identify OSK1 analogs with improved Kv1.3activity and selectivity, upon considering the Kv1.3 and Kv1.1bioactivity information provided in Tables 37 through 42 and thesolution NMR structure of OSK1 described above. FIG. 89C shows a wormrendering of the OSK1 structure with secondary structure elements (betastrands and alpha helices) depicted. The primary amino acid sequence ofOSK1 is provided in FIG. 89E and amino acid residues comprising the betastrands & alpha helix are underlined. Wiggly lines in FIG. 89C indicateamino acid residues between or beyond these secondary structureelements, whereas straight lines depict the three disulfide bridges inOSK1. The first beta strand (β1) shown in FIGS. 89C and 89E contains nodisulfide bridges to link it covalently to other secondary structureelements of the OSK1 molecule, unlike beta strand 3 (β3) that has twodisulfide bridges with the alpha helix (α1). As shown in Table 42, OSK1analogs without beta strand 1 (labeled “des 1-7”) still retain activityin blocking inflammation (see SEQ ID No: 4989 of Table 42) suggestingthat this region of OSK1 is not essential for the molecules Kv1.3bioactivity.

OSK1 analogs containing multiple amino acid changes were generated andtheir activity in the human whole blood assay of inflammation isprovided in Table 42. Several analogs retain high potency in this assaydespite as many as 12 amino acid changes. Based on the improved Kv1.3selectivity of analogs with single amino acid changes, anologs withmultiple amino acid changes may result in additional improvements inselectivity. It is also envisioned that analogs with multiple amino acidchanges may have improved activity or stability in vivo, alone or in thecontext of a peptide conjugate to a half-life prolonging moiety.

Kv1.3 peptide toxins conjugated to half-life prolonging moieties areprovided within this application. The bioactivity of several toxinconjugates is described in Table 43. OSK1 analogs with a N-terminalhalf-life prolonging 20K PEG moiety (see Example 48) were found toprovide potent suppression of the whole blood IL-2 (“WB/IL-2”) and IFNg(“WB/IFNg”) response (Table 43). The 20K PEG-ShK conjugate, shownearlier to have prolonged half-life in vivo (see Examples 44 and 48),was also highly active in this whole blood assay. The FcLoop-OSK1conjugates (see Example 49) were highly active in blocking inflammation(Table 43), and the CH2-OSK1 or PEG-CH2-OSK1 conjugates (see Example 54)provided modest blockade of the whole blood cytokine response (Table43). The IL-2 and IFNg cytokine response measured in this whole bloodassay results from T cell activation. Since this cytokine response isKv1.3 dependent and potently blocked by the Kv1.3 peptide andpeptide-conjugate inhibitors described herein, these whole blood studiesillustrate the therapeutic utility of these molecules in treatment ofimmune disorders.

TABLE 36 Activity of OSK1 analogs in blocking thapsigargin-induced IL-2and IFNg production in 50% human whole blood as described in Example 46.Thapsigargin Induced IL-2 & IFNg in Human Whole Blood Average Std DevAverage Std Dev Analog IC₅₀ Divided IC50 (nM) IC50 (nM) IC50 (nM) IC50(nM) by Ala-1 IC₅₀ OSK1 Analog IL-2 IL-2 IFNg IFNg IL2 IFNg Ala-1 (SEQID No: 1400) 0.1220 0.0791 0.1194 0.0802 1.00 1.00 Ala-2 (SEQ ID No:1401) 0.0884 0.0733 0.1035 0.0776 0.72 0.87 Ala-3 (SEQ ID No: 1402)0.0883 0.0558 0.0992 0.1007 0.72 0.83 Ala-4 (SEQ ID No: 1403) 0.11090.1098 0.0873 0.0993 0.91 0.73 Ala-5 (SEQ ID No: 1404) 0.0679 0.05660.0670 0.0446 0.56 0.56 Ala-6 (SEQ ID No: 1405) 0.0733 0.0477 0.08050.0696 0.60 0.67 Ala-7 (SEQ ID No: 1406) 0.0675 0.0383 0.0591 0.02600.55 0.49 Ala-9 (SEQ ID No: 1407) 0.0796 0.0761 0.0711 0.0627 0.65 0.60Ala-10 (SEQ ID No: 1408) 0.0500 0.0425 0.0296 0.0084 0.41 0.25 Ala-12(SEQ ID No: 1410) 0.1235 0.0823 0.1551 0.0666 1.01 1.30 Ala-13 (SEQ IDNo: 1411) 0.1481 0.0040 0.1328 0.0153 1.21 1.11 Ala-15 (SEQ ID No: 1412)0.1075 0.1075 0.88 0.90 Ala-16 (SEQ ID No: 1413) 0.1009 0.1009 0.83 0.84Ala-17 (SEQ ID No: 1414) 0.1730 0.1730 1.42 1.45 Ala-19 (SEQ ID No:1415) 0.1625 0.1625 1.33 1.36 Ala-20 (SEQ ID No: 1416) 0.3790 0.37903.11 3.17 Ala-22 (SEQ ID No: 1418) 7.0860 7.0860 58.07 59.33 Ala-23 (SEQID No: 1419) 0.2747 0.2747 2.25 2.30 Ala-25 (SEQ ID No: 1421) 3.08003.0800 25.24 25.79 Ala-27 (SEQ ID No: 1423) 3.4510 2.5781 1.5792 1.921720.28 13.22 Ala-29 (SEQ ID No: 1424) 0.4469 0.1727 0.2919 0.2422 3.662.44 Ala-30 (SEQ ID No: 1425) 0.9710 0.7533 0.6370 0.2674 7.96 5.33Ala-34 (SEQ ID No: 1428) 0.0725 0.0275 0.0573 0.0341 0.59 0.48 Pro-12,Lys-16, Asp-20, Ile- 0.5138 0.4064 0.5127 0.1597 4.21 4.29 23, Ile-29,Ala-34 (SEQ ID No: 1393)

TABLE 37 OSK1 Alanine Analogs. SEQ ID Analogue Activity (IC50, pM) NO:Analogue Kv1.3 Kv1.1 WB/IL-2 WB/IFNg 1400 G1A 41.11 13.89 122.035119.425 1401 V2A 81.78 9.94 88.395 103.515 1402 I3A 96.59 10.64 88.25599.16 1403 I4A 195.30 16.92 110.865 87.255 1404 N5A 159.98 14.01 67.9166.985 1405 V6A 173.75 12.84 73.26 80.465 1406 K7A 181.04 21.88 67.559.075 1407 K9A 166.27 40.59 79.58 71.065 1408 I10A 91.23 4.46 49.9729.63 1409 S11A 40.79 113.15 90 110 1410 R12A 389.90 55.89 123.49 155.11411 Q13A 249.46 21.65 148.05 132.75 1412 L15A 43.07 15.04 107.5 107.51413 E16A 21.55 6.87 100.9 100.9 1414 P17A 33.89 9.08 173 173 1415 K19A210.48 16.85 162.5 162.5 1416 K20A 1036.08 185.01 379 379 1417 1418G22A >3000 >3000 7086 7086 1419 M23A 71.39 38.63 274.7 274.7 1420R24A >3000 1890.78 1421 F25A 1486.97 47.30 3080 3080 1422 G26A 710.98733.36 12075 10730 1423 K27A 232.44 >3000 1232 1579.15 1424 M29A59.47 >3333 446.9 291.85 1425 N30A 692.54 >3000 971 637 1426 G31A 70.1761.78 1427 K32A 41.3 34 1428 H34A 19.36 368.41 72.54 57.29 1429 T36A728.4 723.5 1430 P37A 956 849.7 1431 K38A 221 343

TABLE 38 OSK1 Arginine Analogs. SEQ ID Analogue Activity (IC50, pM) NO:Analogue Kv1.3 Kv1.1 WB/IL-2 WB/IFNg 1432 G1R 68.75 9.91 554 991 1433V2R 133.34 25.79 775 986 1434 I3R 19.90 2.47 148 180 1435 I4R 10.41 1.92168 175 1436 N5R 13.62 2.15 95 120 1437 V6R 8.65 2.40 84 115 1438 K7R13.17 <1.52401 78 71 1439 K9R 11.99 2.01 107 77 1440 I10R 11.68 1.73 307474 1441 S11R 16.72 210.05 2118 4070 1442 Q13R 15.34 <1.52401 160 1721443 L15R 13.73 2.16 93 116 1444 E16R 10.36 <1.52401 556 454 1445 P17R10.42 <1.52401 202 355 1446 K19R 12.57 2.41 44 62 1447 K20R 9.85<1.52401 67 83 1448 A21R 14.92 2.61 90 149 1449 G22R 23.74 3.49 292 3491450 M23R 12.34 2.01 182 148 1451 F25R >3333 817.42 25027 30963 1452G26R >3333 >3333 100000 100000 1453 K27R 1492.94 >3333 15088 10659 1454M29R 200.39 1872.11 11680 7677 1455 N30R 18.90 45.71 405 445 1456 G31R22.16 1.59 314 343 1457 K32R 30.83 7.24 28 34 1458 H34R 13.57 4.49 92108 1459 T36R 1308.07 26.55 9697 10050 1460 P37R 13.32 2.01 229 253 1461K38R 14.99 1.84 39 40

TABLE 39 OSK1 Glutamic Acid Analogs. SEQ ID Analogue Activity (IC50, pM)NO: Analogue Kv1.3 Kv1.1 WB/IL-2 WB/IFNg 1462 G1E 185.78 50.97 1217 12521463 V2E 36.23 35.01 97 184 1464 I3E 22.00 42.99 120 160 1465 I4E 15.653.19 218 191 1466 N5E 23.38 4.44 100 65 1467 V6E 17.73 2.43 48 68 1468K7E 14.16 <1.52401 58 68 1469 K9E 31.76 110.67 179 171 1470 I10E 120.3533.50 2573 2736 1471 S11E >3333 >3333 39878 16927 1472 R12E 89.71 193.251787 2001 1473 Q13E 45.87 6.28 1063 799 1474 L15E 47.48 436.05 785 10591475 P17E 14.47 1.81 520 947 1476 K19E 23.51 13.71 1477 K20E 25.45 5.761478 A21E 7.37 <1.52401 117 138 1479 G22E 13.88 2.56 109 164 1480 M23E24.28 10.44 606 666 1481 R24E 7161 9543 1482 F25E >3333 >3333 100000100000 1483 G26E >3333 >3333 100000 100000 1484 K27E >3333 548.55 55487144 1485 M29E >3333 >3333 27099 24646 1486 N30E 14024 24372 1487 G31E12.01 2.37 95 111 1488 K32E 15.56 17.31 62 63 1489 H34E 330.15 1689.821618 2378 1490 T36E 161.06 >3333 1742 2420 1491 P37E 62.67 622.18 2391604 1492 K38E 25.76 34.33 526 713

TABLE 40 OSK1 Naphthylalanine Analogs. SEQ ID Analogue Activity (IC50,pM) NO: Analogue Kv1.3 Kv1.1 WB/IL-2 WB/IFNg 1493 G1Nal 20.66 33.93 27932565 1494 V2Nal 11.55 2.46 750 524 1495 I3Nal 10.31 2.34 907 739 1496I4Nal 15.03 <1.52401 1094 1014 1497 N5Nal 21.78 <1.52401 760 431 1498V6Nal 20.97 <1.52401 1776 2465 1499 K7Nal 23.61 <1.52401 222 246 1500K9Nal 65.82 2.92 1070 1217 1501 I10Nal 45.44 <1.52401 184 257 1502S11Nal 95.87 >3333 23915 17939 1503 R12Nal 37.66 24.99 460 387 1504Q13Nal 13.44 <1.52401 140 198 1505 L15Nal 17.84 <1.52401 358 370 1506E16Nal 9.58 <1.52401 1025 1511 1507 P17Nal 16.19 <1.52401 193 357 1508K19Nal 17.22 <1.52401 58 99 1509 K20Nal 13.53 <1.52401 74 125 1510A21Nal 26.10 <1.52401 315 434 1511 G22Nal >3333 426.27 10328 10627 1512M23Nal 35.96 64.37 581 1113 1513 R24Nal 45.26 2.85 293 818 1514 F25Nal28.63 51.75 1733 1686 1515 G26Nal >3333 1573.75 9898 10651 1516K27Nal >3333 1042.84 14971 27025 1517 M29Nal 93.46 46.88 100000 1000001518 N30Nal >3333 1283.88 100000 37043 1519 G31Nal 33.76 <1.52401 331467 1520 K32Nal 26.13 1.91 134 196 1521 H34Nal 60.31 >3333 3323 61861522 T36Nal 100000 37811 1523 P37Nal 70.80 6.74 1762 3037 1524 K38Nal308 409

TABLE 41 OSK1 Analogues with Improved Selectivity at Kv1.3 over Kv1.1(whole cell patch clamp ePhys). SEQ Kv1.3 Kv1.1 Kv1.3 Selectivity ID NO:Analogue (IC50, pM) (IC50, pM) (= Kv1.1/Kv.3 IC50) 25 wild-type 39  2025 1441 S11R 40  9130 228 1502 S11Nal 1490 85324 57 1410 R12A 25  440 171474 L15E 190 65014 342 1423 K27A 289  10085* 35 1424 M29A 33  3472 1051454 M29R 760 23028 30 1425 N30A 766 10168 14 1428 H34A 16 12754 7971521 H34Nal 215 29178 136 1489 H34E 1322 39352 30 1490 T36E 1921 8391444 1491 P37E 241 15699 65 *PatchXpress data

TABLE 42 OSK1 Analogues with Multiple Amino Acid Substitutions. # AminoAcid WB/IL-2 WB/IFNg SEQ ID NO: Changes Amino Acid Changes (IC50, nM)(IC50, nM) 4988 2 M29A, H34Nal 0.087 0.102 296 2 E16K, K20D 1.579 1.324986 2 I4K(Gly), H34A 0.470 0.451 4987 2 H34A, K38K(Gly) 1.249 2.3804985 2 K19K(Gly), H34A 1.514 1.633 1392 3 R12A, E16K, K20D 0.041 0.0924990 3 E16K, K20D, H34A 65.462 26.629 298 3 E16K, K20D, T36Y 0.639 0.9234991 4 S11Nal, R12A, M29A, H34Nal 12.665 14.151 1396 5 E16K, K20D,des36-38 3.941 6.988 1395 5 GGGGS-Osk1 1.357 2.204 1274 5 E16K, K20D,T36G, P37G, K38G 2.636 3.639 4992 5 S11Nal, R12A, M29A, H34Nal, P37E3.511 5.728 4994 5 S11Nal, R12A, M23F, M29A, H34Nal 8.136 22.727 1398 5R12P, E16K, K20D, T37Y, K38Ne 0.527 0.736 1397 5 R12P, E16Om, K20E,T37Y, K38Ne 6.611 18.454 4995 6 S11Nal, R12A M23Ne, M29A, H34Nal, P37E14.32 68.158 4916 7 des1, V2G, R12A, E16K, K19R, K20D, H34A 1.499 2.2444993 7 S11Nal, R12A, L15E, M29A, H34Nal, T36E, P37E >100 >100 4989 12des1-7, E16K, K20D, des36-38 8.179 8.341

TABLE 43 Bioactivity of OSK1 and OSK1 peptide analog conjugates withhalf-life-extending moieties as indicated. Fcloop structures G2-OSK1-G2(SEQ ID NO:976), G4-OSK1-G2 (SEQ ID NO:979), and G2-ShK-G2 (SEQ IDNO:977) are described in Example 49, and CH2-L10-OSK1(1-38) SEQ IDNO:4917 is described in Example 54. F¹ (and WB/IL-2 WB/IFNg F², if(IC50, (IC50, present) Short-hand Designation nM) nM) PEG 20kPEG-OSK1[Ala12] 0.270 0.137 PEG 20k PEG-OSK1[Ala29] 5.756 5.577 PEG 20kPEG-OSK1[Ala29, 1Nal34] 0.049 0.081 PEG 20k PEG-OSK1[1Nal11] 0.019 0.027PEG 20k PEG-ShK 0.046 0.065 FcLoop FcLoop-G2-OSK-G2 0.028 0.056 FcLoopFcLoop-G4-OSK-G2 0.150 0.195 FcLoop FcLoop-G2-ShK-G2 0.109 0.119 PEG-20k PEG-L2-6H-L3-CH2-L10- 8.325 50.144 CH2 OsK1(1-38) CH2L2-6H-L3-CH2-L10-OsK1(1-38) 38.491 55.162

Example 56 Design and Expression of Monovalent Fc-Fusion Molecules

There may be pharmacokinetic or other reasons, in some cases, to prefera monovalent dimeric Fc-toxin peptide fusion (as representedschematically in FIG. 2B) to a (“bivalent”) dimer (as representedschematically in FIG. 2C). However, conventional Fc fusion constructstypically result in a mixture containing predominantly dimericmolecules, both monovalent and bivalent. Monovalent dimeric Fc-toxinpeptide fusions (or “peptibodies”), including monovalent dimeric Fc-OSK1peptide analog fusions and Fc-ShK peptide analog fusions, can beisolated from conditioned media which also contains bivalent dimericFc-toxin peptide, and dimeric Fc lacking the toxin peptide fusion.Separation of all three species can be accomplished using ion exchangechromatography, for example, as described in Examples 1, 2, and 41herein.

A number of other exemplary ways that a monovalent dimeric Fc-toxinpeptide fusion can be produced with greater efficiency are providedhere, including for the production of monovalent dimeric Fc-OSK1 peptideanalog fusions:

(1) Co-expressing equal amounts of Fc and Fc-toxin peptide in the samecells (e.g. mammalian cells). With the appropriate design, a mixture ofbivalent dimeric Fc-toxin peptide fusion, monovalent dimeric Fc-toxinpeptide fusion and dimeric Fc will be produced and released into theconditioned media. The monovalent dimeric Fc-toxin peptide can bepurified from the mixture using conventional purification methods, forexample, methods described in Examples 1, 2, and 41 herein.

(2) Engineering and recombinantly expressing in mammalian cells a singlepolypeptide construct represented by the following schematic:Signal peptide-Fc-furin cleavage site-linker-furin cleavagesite-Fc-toxin peptideFurin cleavage occurs as the molecule travels through the endoplasmicreticulum and the intra-molecular Fc pairing (resulting in monovalentdimeric Fc-toxin peptide fusion) can occur preferentially tointermolecular Fc pairing (resulting in dimeric Fc-toxin peptide beingexpressed into conditioned medium; FIG. 87A-B).

By way of example of method (2) above, a DNA construct was produced forrecombinant expression in mammalian cells of the following schematicpolypeptide construct:Signal peptide-Fc-furin cleavage site-linker-furin cleavagesite-Fc-ShK(2-35)

The DNA construct had the following nucleotide coding sequence:

//SEQ ID NO: 5007 atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaacgaggcaagagggctgtggggggcggtgggagcggcggcgggggctcaggtggcgggggaagtggcgggggagggagtggagggggagggagtggaggcgggggatccggcggggggggtagcaagcgtcgcgagaagcgggataagacccatacctgccccccctgtcccgcgcccgagttgctcgggggccccagcgtgtttttgtttcctcccaagcctaaagatacattgatgattagtagaacacccgaagtgacctgtgtcgtcgtcgatgtctctcatgaggatcccgaagtgaaattcaattggtatgtcgatggggtcgaagtccacaacgctaaaaccaaacccagagaagaacagtataattctacctatagggtcgtgtctgtgttgacagtgctccatcaagattggctcaacgggaaagaatacaaatgtaaagtgagtaataaggctttgcccgctcctattgaaaagacaattagtaaggctaagggccaacctagggagccccaagtctatacactccctcccagtagagacgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaaggaggaggaggatccggaggaggaggaagcagctgcatcgacaccatccccaagagccgctgcaccgccttccagtgcaagcacagcatgaagtaccgcctgagcttctgccgcaagacctgcggcacctgctaa.

The resulting expressed polypeptide (from vectorpTT5-Fc-Fc-L10-Shk(2-35)) had the following amino acid sequence beforefurin cleavage (the first 19 residues are a signal peptide sequence;furin cleavage sites are underlined):

//SEQ ID NO: 5008 mewswvflfflsvttgvhsdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkrgkravggggsggggsggggsggggsggggsggggsggggskrrekrdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkggggsggggsscidtipksrctafqckhsmk yrlsfcrktcgtc.

FIG. 87A-B demonstrates recombinant expression of a monovalent dimericFc-L-ShK(2-35) molecule product expressed by and released into theconditioned media from transiently transfected mammalian cells. FIG. 88shows results from a pharmacokinetic study on the monovalent dimericFc-ShK(1-35) in SD rats. Serum samples were added to microtiter platescoated with an anti-human Fc antibody to enable affinity capture. Plateswere then washed, captured samples were released by SDS and run on apolyacrylamide gel. Samples were then visualized by western blot usingan anti-human Fc-specific antibody and secondary-HRP conjugate. The MWof bands from serum samples is roughly identical to the originalpurified material, suggesting little, if any, degradation of the proteinoccurred in vivo over a pro-longed half-life, in spite of the presenceof Arg at position 1 of the ShK(1-35) sequence.

(3) Similar to (2) above, a Fc-toxin peptide fusion monomer can beconjugated with an immunoglobulin light chain and heavy chain resultingin a monovalent chimeric immunoglobulin-Fc-toxin peptide molecule. Wehave termed an immunoglobulin (light chain+heavy chain)-Fc construct a“hemibody”; such “hemibodies” containing a dimeric Fc portion canprovide the long half-life typical of a dimeric antibody. The schematicrepresentation in FIG. 92A-C illustrates an embodiment of ahemibody-toxin peptide fusion protein and its recombinant expression bymammalian cells.

If the antibody chosen is a target specific antibody (e.g., ananti-Kv1.3 or anti-IKCa1 antibody), the chimeric molecule may alsoenhance the targeting efficiency of the toxin peptide. FIG. 91A-Bdemonstrates that such chimeric molecules, in this exampleFc-L10-ShK(2-35) dimerized with human IgG1 or human IgG2 light and heavychains, can be expressed and released into the conditioned media fromtransfected mammalian cells.

Example 57 Osk1 PEGylated at Residue 4 by Oxime Formation

[Dp^((AOA)-PEG))4]Osk1 Peptide Synthesis of reduced [Dpr^((AOA))4]Osk1.[Dpr^((AOA))4]Osk1, having the sequence:

(SEQ ID NO: 5009) GVI[Dpr(AOA)]NVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPKcan be synthesized in a stepwise manner on a Symphony™ multi-peptidesynthesizer by solid-phase peptide synthesis (SPPS) using2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU)/N-methyl morpholine (NMM)/N,N-dimethyl-formamide (DMF) couplingchemistry at 0.1 mmol equivalent resin scale on Fmoc-Lys(Boc)-Wang resin(Novabiochem). N-alpha-(9-fluorenylmethyloxycarbonyl)- and side-chainprotected amino acids can be purchased from Novabiochem. The followingside-chain protection strategy can be employed: Asp(O^(t)Bu), Arg(Pbf),Cys(Trt), Gln(Trt), His(Trt), Lys(N^(ε)-Boc), Ser(O^(t)Bu), Thr(O^(t)Bu)and Tyr(O^(t)Bu). Dpr(AOA), i.e.,N-α-Fmoc-N-b-(N-t.-Boc-amino-oxyacetyl)-L-diaminopropionic acid, can bepurchased from Novabiochem (Cat. No. 04-12-1185). The protected aminoacid derivatives (20 mmol) can be dissolved in 100 ml 20% dimethylsulfoxide (DMSO) in DMF (v/v). Protected amino acids can be activatedwith 200 mM HBTU, 400 mM NMM in 20% DMSO in DMF, and coupling can becarried out using two treatments with 0.5 mmol protected amino acid, 0.5mmol HBTU, 1 mmol NMM in 20% DMF/DMSO for 25 minutes and then 40minutes. Fmoc deprotection reactions can be carried out with twotreatments using a 20% piperidine in DMF (v/v) solution for 10 minutesthen 15 minutes. Following synthesis and removal of the N-terminal Fmocgroup, the resin can be then drained, and washed with DCM, DMF, DCM, andthen dried in vacuo. The peptide-resin can be deprotected and releasedfrom the resin by treatment with a TFA/amionooxyacetic acid/TIS/EDT/H2O(90:2.5:2.5:2.5:2.5) solution at room temperature for 1 hour. Thevolatiles can be then removed with a stream of nitrogen gas, the crudepeptide precipitated twice with cold diethyl ether and collected bycentrifugation. The [Dpr^((AOA))4]Osk1 peptide can be then analyzed on aWaters 2795 analytical RP-HPLC system using a linear gradient (0-60%buffer B in 12 minutes, A: 0.1% TFA in water also containing 0.1%aminooxyacetic acid, B: 0.1% TFA in acetonitrile) on a Jupiter 4 μmProteo™ 90 Å column.

Reversed-Phase HPLC Purification. Preparative Reversed-phasehigh-performance liquid chromatography can be performed on C18, 5 μm,2.2 cm×25 cm) column. The [Dpr^((AOA))4]Osk1 peptide is dissolved in 50%aqueous acetronitrile containing acetic acid and amionooxyacetic acidand loaded onto a preparative HPLC column. Chromatographic separationscan be achieved using linear gradients of buffer B in A (A=0.1% aqueousTFA; B=90% aq. ACN containing 0.09% TFA), typically 5-95% over 90minutes at 15 mL/min. Preparative HPLC fractions can be characterized byESMS and photodiode array (PDA) HPLC, combined and lyophilized.

Osk1 peptide analog PEGylated at residue 4 by oxime formation:[Dpr^((AOA)-PEG))4]Osk1 (i.e.,GVI[Dpr^((AOA-PEG))]NVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK//SEQ ID NO:5010)can be made as follows. Lyophilized [Dpr^((AOA))4]Osk1 peptide can bedissolved in 50% HPLC buffer A/B (5 mg/mL) and added to a two-fold molarexcess of MeO-PEG-aldehyde, CH₃O—[CH₂CH₂O]_(n)—CH₂CH₂CHO (averagemolecular weight 20 kDa). The aminoxyacetyl group within the peptide atresidue 4 reacts with the aldehyde group of the PEG to form a covalentoxime linkage. The reaction can be left for 24 hours, and can beanalyzed on an Agilent™ 1100 RP-HPLC system using Zorbax™ 300SB-C8 5 μmcolumn at 40° C. with a linear gradient (6-60% B in 16 minutes, A: 0.1%TFA in water, B: 0.1% TFA/90% ACN in water). Mono PEGylated[Dpr^((AOA)-PEG))4]Osk1 peptide can be then isolated using a HiTrap™ 5mL SP HP cation exchange column on AKTA FPLC system at 4° C. at 1 mL/minusing a gradient of 0-50% B in 25 column volumes (Buffers: A=20 mMsodium acetate pH 4.0, B=1 M NaCl, 20 mM sodium acetate, pH 4.0). Thefractions can be analyzed using a 4-20 tris-Gly SDS-PAGE gel andRP-HPLC. SDS-PAGE gels can be run for 1.5 hours at 125 V, 35 mA, 5 W.Pooled product, mono-PEGylated [Dpr^((AOA)-PEG))4]Osk1 peptide, can bethen dialyzed at 4° C. in 3 changes of 1 L of A4S buffer (10 mM NaOAc,5% sorbitol, pH 4.0). The dialyzed product can be then concentrated in10 K microcentrifuge filter to 2 mL volume and sterile-filtered using0.2 μM syringe filter to give the final product.

Folding of [Dpr^((AOA)-PEG))4]Osk1 (Disulphide bond formation). Themono-PEGylated [Dpr^((AOA)-PEG))4]Osk1 peptide can be dissolved in 20%AcOH in water (v/v) and can be then diluted with water to aconcentration of approximately 0.15 mg peptide mL, the pH adjusted toabout 8.0 using NH₄OH (28-30%), and gently stirred at room temperaturefor 36 hours. Folding process can be monitored by LC-MS analysis.Following this, folded mono-PEGylated [Dpr^((AOA)-PEG))4]Osk1 can bepurified using by reversed phase HPLC using a 1″ Luna 5 μm C18 100 ÅProteo™ column with a linear gradient 0-40% buffer B in 120 min (A=0.1%TFA in water, B=0.1% TFA in acetonitrile). Mono-PEGylated (oximated)[Dpr^((AOA)-PEG))4]Osk1 peptide disulfide connectivity can be C1-C4,C2-C5, and C3-C6.

ABBREVIATIONS

Abbreviations used throughout this specification are as defined below,unless otherwise defined in specific circumstances.

Ac acetyl (used to refer to acetylated residues)

AcBpa acetylated p-benzoyl-L-phenylalanine

ADCC antibody-dependent cellular cytotoxicity

Aib aminoisobutyric acid

bA beta-alanine

Bpa p-benzoyl-L-phenylalanine

BrAc bromoacetyl (BrCH₂C(O)

BSA Bovine serum albumin

Bzl Benzyl

Cap Caproic acid

COPD Chronic obstructive pulmonary disease

CTL Cytotoxic T lymphocytes

DCC Dicylcohexylcarbodiimide

Dde 1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)ethyl

ESI-MS Electron spray ionization mass spectrometry

Fmoc fluorenylmethoxycarbonyl

HOBt 1-Hydroxybenzotriazole

HPLC high performance liquid chromatography

HSL homoserine lactone

IB inclusion bodies

KCa calcium-activated potassium channel (including IKCa, BKCa, SKCa)

Kv voltage-gated potassium channel

Lau Lauric acid

LPS lipopolysaccharide

LYMPH lymphocytes

MALDI-MS Matrix-assisted laser desorption ionization mass spectrometry

Me methyl

MeO methoxy

MHC major histocompatibility complex

MMP matrix metalloproteinase

1-Nap 1-napthylalanine

NEUT neutrophils

Nle norleucine

NMP N-methyl-2-pyrrolidinone

PAGE polyacrylamide gel electrophoresis

PBMC peripheral blood mononuclear cell

PBS Phosphate-buffered saline

Pbf 2,2,4,6,7-pendamethyldihydrobenzofuran-5-sulfonyl

PCR polymerase chain reaction

Pec pipecolic acid

PEG Poly(ethylene glycol)

pGlu pyroglutamic acid

Pic picolinic acid

pY phosphotyrosine

RBS ribosome binding site

RT room temperature (25° C.)

Sar sarcosine

SDS sodium dodecyl sulfate

STK serine-threonine kinases

t-Boc tert-Butoxycarbonyl

tBu tert-Butyl

THF thymic humoral factor

Trt trityl

1. A composition of matter of the formula(X¹)_(a)—(F¹)_(d)—(X²)_(b)—(F²)_(e)—(X³)_(c) and multimers thereof,wherein: F¹ and F² are half-life extending moieties, wherein F¹ or F²,or both is a polyethylene glycol, a copolymer of ethylene glycol, apolypropylene glycol, a copolymer of propylene glycol, acarboxymethylcellulose, a polyvinyl pyrrolidone, a poly-1,3-dioxolane ,a poly-1,3,6-trioxane, an ethylene/maleic anhydride copolymer, apolyaminoacid, a dextran n-vinyl pyrrolidone, a poly n-vinylpyrrolidone, a propylene glycol homopolymer, a propylene oxide polymer,an ethylene oxide polymer, a polyoxyethylated polyol, a polyvinylalcohol, a linear or branched glycosylated chain, a polyacetal, a longchain fatty acid, a long chain hydrophobic aliphatic group, animmunoglobulin light chain and heavy chain, an immunoglobulin F_(c)domain or portion thereof, a CH2 domain of Fc, an Fc domain loop, analbumin, an albumin-binding protein, a transthyretin, athyroxine-binding globulin, or a ligand that has an affinity for a longhalf-life serum protein, said ligand being selected from the groupconsisting of peptide ligands and small molecule ligands; or acombination of any of these members; and d and e are each independently0 or 1, provided that at least one of d and e is 1; X¹, X², and X³ areeach independently -(L)_(f)-P-(L)_(g), and f and g are eachindependently 0 or 1; P is a toxin peptide of no more than about 80amino acid residues in length, comprising at least two intrapeptidedisulfide bonds, and at least one P is an OSK1 peptide analog comprisingthe amino acid sequence of SEQ ID NO:4916; L is a linker; and a, b, andc are each independently 0 or 1, provided that at least one of a, b andc is
 1. 2. The composition of matter of claim 1 of the formulaP-(L)_(g)-F¹.
 3. The composition of matter of claim 1 of the formulaF¹-(L)_(f)-P.
 4. The composition of matter of claim 1 of the formulaP-(L)_(g)-F¹-(L) _(f)-P.
 5. The composition of matter of claim 1 of theformula F¹-(L)_(f)-P-(L) _(g)-F².
 6. The composition of matter of claim1 of the formula F¹-(L)_(f)-P-(L)_(g)F ²-(L)_(f)-P.
 7. The compositionof matter of claim 1 of the formula F¹-F²-(L)_(f)-P.
 8. The compositionof matter of claim 1 of the formula P-(L)_(g)-F¹-F².
 9. The compositionof matter of claim 1 of the formula P-(L)_(g)-F¹-F²-(L) _(f)-P.
 10. Thecomposition of matter of claim 1 wherein F¹ or F², or both, comprises ahuman IgG Fc domain or a portion thereof.
 11. The composition of matterof claim 1, wherein F¹ and F² are different half-life extendingmoieties.
 12. The composition of matter of claim 1, wherein F¹ or F², orboth, comprises a sequence selected from SEQ ID NOS: 2, 4, 70, 71, 72,74, 75, 76, 1340 through 1342, and 1359 through 1363 as set forth inFIGS. 3, 4, 11A-C, 12A-C, and 12E-F.
 13. The composition of matter ofclaim 1, wherein F¹ or F², or both, comprises a biologically suitablepolymer or copolymer.
 14. The composition of matter of claim 1, in whichthe toxin peptide is inserted into a human IgG1 Fc domain loop.
 15. Thecomposition of matter of claim 1, wherein the C-terminal carboxylic acidmoiety of the OSK1 peptide analog is replaced with a moiety selectedfrom (A) —COOR, where R is independently (C₁-C₈)alkyl, haloalkyl, arylor heteroaryl; (B) —C(═O)NRR, where R is independently hydrogen,(C₁-C₈)alkyl, haloalkyl, aryl or heteroaryl; and (C) —CH₂OR where R ishydrogen, (C₁-C₈) alkyl, aryl or heteroaryl.
 16. The composition ofmatter of claim 1, wherein the OSK1 peptide analog is conjugated to apolyethylene glycol (PEG) via: (a) 1, 2, 3 or 4 amino functionalizedsites of the PEG; (b) 1, 2, 3 or 4 thiol functionalized sites of thePEG; (c) 1, 2, 3 or 4 maleimido functionalized sites of the PEG; (d) 1,2, 3 or 4 N-succinimidyl functionalized sites of the PEG; (e) 1, 2, 3 or4 carboxyl functionalized sites of the PEG; or (f) 1, 2, 3 or 4p-nitrophenyloxycarbonyl functionalized sites of the PEG.
 17. Thecomposition of matter of claim 1, wherein the OSK1 peptide analog isconjugated to an acyl, aryl, fatty acid, or polyethylene glycol (PEG)via: (a) 1, 2, 3 or 4 amino functionalized sites in the OSK1 peptideanalog; (b) 1, 2, 3 or 4 thiol functionalized sites in the OSK1 peptideanalog; (c) 1 or 2 ketone functionalized sites in the OSK1 peptideanalog; (d) 1 or 2 azido functionalized sites in the OSK1 peptideanalog; (e) 1 or 2 carboxyl functionalized sites in the OSK1 peptideanalog; (f) 1 or 2 aminooxy functionalized sites in the OSK1 peptideanalog; or (g) 1 or 2 seleno functionalized sites in the OSK1 peptideanalog.
 18. The composition of matter of claim 1, wherein the OSK1peptide analog is covalently linked at its N-terminal to a moietyselected from acyl, aryl, fatty acid, or polyethylene glycol.
 19. Thecomposition of matter of claim 1, wherein the OSK1 peptide analog iscovalently linked at its N-terminal to a moiety selected from benzyl,dibenzyl, benzoyl, benzyloxycarbonyl, N,N-dimethylglycine, creatine,formyl, acetyl, propanoyl, butanyl, heptanyl, hexanoyl, octanoyl,nonanoyl, butyric acid, caproic acid, caprylic acid, capric acid, lauricacid, myristic acid, palmitic acid, and stearic acid.
 20. Thecomposition of matter claim 1, further comprising, covalently bound toF1, F2, or to P, an additional agonistic peptide or an antagonisticpeptide, in relation to the activity of the OSK1 peptide analog, or atargeting peptide.
 21. A pharmaceutical composition, comprising thecomposition of claim 1 and a pharmaceutically acceptable carrier.
 22. Acomposition of matter of the formula(X¹)_(a)—(F¹)_(d)—(X²)_(b)—(F²)_(e)—(X³)_(c) and multimers thereof,wherein: F¹ and F² are half-life extending moieties selected from anacyl, aryl, fatty acid, and polyethylene glycol (PEG), and d and e areeach independently 0 or 1, provided that at least one of d and e is 1;X¹, X², and X³ are each independently -(L)_(f)-P-(L)_(g)-, and f and gare each independently 0 or 1; P is an OSK1 peptide analog of no morethan about 80 amino acid residues in length, comprising at least twointrapeptide disulfide bonds, comprising the amino acid sequence of SEQID NO:4916; L is a linker; a, b, and c are each independently 0 or 1,provided that at least one of a, b and c is 1; and the toxin peptide isconjugated to F¹ or F², or both, via: (a) 1, 2, 3 or 4 aminofunctionalized sites in the toxin peptide; (b) 1, 2, 3 or 4 thiolfunctionalized sites in the toxin peptide; (c) 1 or 2 ketonefunctionalized sites in the toxin peptide; (d) 1 or 2 azidofunctionalized sites in the toxin peptide; (e) 1 or 2 carboxylfunctionalized sites in the toxin peptide; (f) 1 or 2 aminooxyfunctionalized sites in the toxin peptide; or (g) 1 or 2 selenofunctionalized sites in the toxin peptide.
 23. A pharmaceuticalcomposition, comprising the composition of claim 22 and apharmaceutically acceptable carrier.